Enzyme substrate comprising a functional dye and associated technology and methods

ABSTRACT

Enzyme substrates and associated technology of the present invention are provided. An enzyme substrate of the invention may comprise a biologically functional fluorescent dye and an enzyme-specific substrate moiety attached in such a way that the functionality of the functional dye is diminished. An enzymatic reaction may cleave at least a portion of the substrate moiety from the enzyme substrate to provide a more functional product dye. This product dye may be nonfluorescent or weakly fluorescent, in general, and relatively fluorescent, in a particular condition, such as when bound to a partner biological molecule or an assembly of partner biological molecules. An enzyme substrate of the present invention may thus be useful in fluorescence detection, and/or in any of a variety of useful applications, such as the detection of enzymatic activity in a cell-free system or in a living cell, the screening of drugs, or the diagnosis of disease.

REFERENCE TO A SEQUENCE LISTING SUBMITTED IN ELECTRONIC FORMAT IN A TEXTFILE AND INCORPORATION BY REFERENCE OF THE CONTENTS THE TEXT FILE

A text file, containing the Sequence Listing for SEQ ID NO: 1 throughSEQ ID NO: 13 disclosed herein, is electronically submitted herewith,hereby referred to herein, and hereby incorporated herein in itsentirety, including the contents thereof, by this reference.

BACKGROUND

Fluorescent substances or fluorogenic substances, such as those that areresponsive to enzyme activity, have a variety of useful applications.Such substances have been used in biological assays, for example.

Enzyme activity in a biological sample, such as a cell, a cell extract,a tissue sample, a biological fluid, a whole organism, and/or the like,is often associated with cellular metabolism, disease state, the successof a genetic manipulation, the identity of a particular microorganism,and/or the like. The ability to detect enzyme activity in a sensitiveand quantitative manner may be useful for any of a variety ofapplications, such as use in cell biology, disease diagnosis,identification of a biological toxin, drug screening, and/or the like,for example. One way to detect enzyme activity is through the use of afluorogenic enzyme substrate, which is a generally nonfluorescent oronly weakly fluorescent compound until it is enzymatically cleaved torelease a highly fluorescent dye.

Traditionally, a fluorogenic enzyme substrate has been produced bycovalently linking a functional group of a fluorescent dye to asubstrate moiety molecule, where the substrate moiety molecule mimicsthe natural enzyme substrate and thus is recognized by the enzyme beinginvestigated. In general, the functional group of the dye, which istypically either an aromatic primary amine or an aromatic hydroxy group,is an integral part of the chromophoric core structure of the dye, inwhich the presence of the functional group imparts a spectral propertyor spectral properties unique to the dye. When the functional group ofthe dye is covalently linked to a substrate moiety molecule, thefunctionality of the functional group is changed, resulting in adramatic blue shift in the absorption and emission wavelengths of thedye and a concomitant reduction in the fluorescent quantum yield of thedye. In some cases, the covalent linkage between the functional groupand the substrate moiety molecule of the dye results in a completelycolorless and non-fluorescent enzyme substrate. In general, when thefunctional group of the dye is an aromatic primary amine group, thesubstrate moiety molecule is an amino acid or a peptide that isrecognized by a peptidase. In general, when the functional group of thedye is a hydroxy group, the substrate moiety molecule may be any of avariety of substrate moiety molecules, such as a glycosidyl that isrecognized by a glycosidase, a phosphoryl that is recognized by aphosphatase, an alkyl that is recognized by a cytochrome P450 enzyme, oran acyl that is recognized by an esterase, for example. Enzymatichydrolysis of the fluorogenic enzyme substrate cleaves the bond betweenthe dye and the substrate moiety molecule, thus regenerating thefluorescent dye at a rate proportional to the level of enzyme activity.

Fluorogenic enzyme substrates have been produced using any of a numberof fluorescent dyes. For example, amine-containing dyes, such asrhodamine 110, 7-amino-4-methylcoumarin, and7-amino-4-trifluoromethylcoumarin, for example, have been used forpreparing fluorogenic peptidase substrates. Further by way of example,hydroxy-containing dyes, such as fluorescein,7-hydroxy-4-methylcoumarin, and resorufin, for example, have been usedfor preparing fluorogenic enzyme substrates in which the enzyme cleavagesite is a bond between an oxygen atom and the enzyme substrate moietymolecule. In Table 1 below, a list of a few dyes that have been used forconstructing fluorogenic enzyme substrates is provided, along withidentifications of the functional group associated with each dye, thesubstrate moiety molecule associated with each dye, the type of linkagebetween the functional group and the substrate moiety moleculeassociated with each dye, and the enzyme that corresponds to thefluorogenic enzyme substrate associated with each dye, merely by way ofexample.

TABLE 1 Dyes, and Associated Functional Groups, Substrate MoietyMolecules, Linkages, and Enzymes Functional Substrate Moiety Dye GroupMolecule Linkage Enzyme rhodamine 110 amine amino acid or amide bondpeptidase peptide 7-amino-4- amine amino acid or amide bond peptidasemethylcoumarin peptide Fluorescein hydroxy carboxylic acid ester bondesterase Fluorescein hydroxy β-D-galactose ether bond β- galactosidaseFluorescein hydroxy α-D-glucose ether bond α-glucosidase Fluoresceinhydroxy β-D-cellobiose ether bond cellulase Fluorescein hydroxyphosphate phosphoester bond phosphatase Resorufin hydroxy alkyl etherbond cytochrome P450 7-hydroxy-4- hydroxy sulfate sulfoester arylsulfatase methylcoumarin bond

Fluorogenic enzyme substrates have also been designed based on theprinciple of fluorescence resonance energy transfer (FRET). SuchFRET-based design has primarily been used for preparing a fluorogenicpeptidase substrate in which the enzyme must bind to both sides of thecleavage site for the enzymatic hydrolysis to take place. A FRET-basedpeptidase substrate has one dye, called the fluorescence donor, attachedto one end of the peptide, and another dye, called the fluorescenceacceptor or the fluorescence quencher, attached to the other end of thepeptide. Prior to the enzymatic cleavage of the substrate, thefluorescence of the donor is substantially quenched by the quencher as aresult of the physical proximity of the donor and quencher. Followingthe enzymatic hydrolysis of the peptide, the donor and quencher areseparated, releasing the fluorescence of the donor at a rateproportional to the level of enzyme activity. There are various examplesof FRET-based peptidase substrates, such as the HIV protease substratedescribed by Wang et al., Tetrahedron Lett. 31, 6493 (1990), the reninsubstrate described by Paschalidou et al., Biochem. J. 382, 1031 (2004),and the HCV substrate by Taliani et al., Anal. Biochem. 240, 60 (1996),for example. All of these FRET-based enzyme substrates employ a bluefluorescent donor dye. A dye having a short wavelength, such as a bluefluorescent dye, for example, is in general not desirable.

A class of fluorogenic substrates for TEM-1 β-lactamase or Bla, which isa bacterial enzyme that catalyzes the breakdown of cephalosporins withhigh efficiency, has been a useful variation of fluorogenic peptidasesubstrates. The gene that encodes for Bla has been used as a reportergene for studying gene expression in eukaryotic cells. Severalfluorogenic Bla substrates have been developed for detecting thereporter enzyme in transfected living cells. One Bla fluorogenicsubstrate, called CCF2, is a FRET-based compound consisting of a donor7-hydroxycoumarin linked via a cephalosporin to an acceptor fluorescein.CCF2 is green fluorescent due to FRET from the donor to the acceptor,but becomes blue fluorescent when hydrolysis of the cephalosporin ringstructure causes the elimination of the fluorescein molecule. (Zlokarniket al., Science 279, 84 (1998).) FRET-based Bla substrates usually haverelatively large molecular weights and poor water solubility, both ofwhich make the substrates difficult to apply to mammalian tissues orcells with thick walls, such as yeast or plant cells, for example. Newfluorogenic Bla substrates have been developed by attaching only asingle dye with a phenolic group to the 3′-position of a cephalosporin.(Gao et al., J. Am. Chem. Soc. 125, 11146 (2003).) Enzymatic hydrolysisof the substrate releases the dye, resulting in a fluorescence increase.These new enzyme substrates have relatively small molecular weights andthus readily enter cells. However, the enzymatically released dyes fromthese single-dye substrates usually lack the ability to be retained inthe cells, making it difficult to identify enzyme-activity-specificcells.

Further development of fluorescent or fluorogenic substances or themaking or the use thereof is desirable.

BRIEF SUMMARY

The present invention provides enzyme substrates and associatedtechnology, including associated systems, kits, methods, and the like.An enzyme substrate of the invention may comprise a biologicallyfunctional fluorescent dye and an enzyme-specific substrate moietyattached in such a way that the functionality of the functional dye isdiminished. An enzymatic reaction may cleave at least a portion of thesubstrate moiety from the enzyme substrate to provide a more functionalproduct dye. This product dye may be nonfluorescent or weaklyfluorescent, in general, and relatively fluorescent, in a particularcondition, such as when bound to a partner molecule, partner molecules,or an assembly of partner molecules. An enzyme substrate of the presentinvention may thus be useful in fluorescence detection, and/or in any ofa variety of useful applications, such as the detection of enzymaticactivity in a cell-free system or in a living cell, the screening ofdrugs, or the diagnosis of disease.

These and various other aspects, features, and embodiments of thepresent invention are further described herein. By this reference, thisbrief summary fully incorporates the sequence listing, any usefulbackground, the figures, the tables, the panels, the descriptions, thestructural formulas, the claims, and the abstract, to the extent samemay be suitable for a summary of subject matter herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of various aspects, features and embodiments ofthe present invention is provided herein with reference to theaccompanying drawings, which are briefly described below. The drawingsare illustrative and are not necessarily drawn to scale. The drawingsillustrate various aspects or features of the present invention and mayillustrate one or more embodiment(s) or example(s) of the presentinvention in whole or in part. A reference numeral, letter, and/orsymbol that is used in one drawing to refer to a particular element orfeature may be used in another drawing to refer to a like element orfeature.

FIG. 1 is a graphical representation of relative florescence emissionintensity versus wavelength (nm), or emission spectra, of the an enzymesubstrate (Substrate No. 20, at 1 μM) and a control compound (CompoundNo. 24, at 1 μM) in the absence or presence of dsDNA (at 35 μg/mL in TEbuffer), with excitation wavelength set at 600 nm and emissioncollection wavelength set at 660 nm, as further described in connectionwith Examples 22, 27 and 51. FIG. 1 also includes an inset graphicalrepresentation of a portion of the emission spectra just described,namely, the boxed-in portion, in an enlarged form.

FIG. 2A is a graphical representation of relative fluorescence versusdsDNA concentration (μg/mL), or a DNA titration, of a nucleic aciddye-based substrate (Substrate No. 19, at 1 μM in TE buffer) for acaspase-3 enzyme and a control compound (Compound No. 7, at 1 μM in TEbuffer), as further described in connection with Examples 7, 10 and 52.

FIG. 2B is a graphical representation of relative fluorescence versusdsDNA concentration (μg/mL), or a DNA titration, of a nucleic aciddye-based substrate (Substrate No. 20, at 1 μM in TE buffer) for acaspase-3 enzyme and a control compound (Compound No. 24, at 1 μM in TEbuffer), as further described in connection with Examples 19, 27 and 52.FIG. 2A and FIG. 2B may be collectively referred to herein as FIG. 2.

FIG. 3 is a graphical representation of relative fluorescence versustime (minutes) associated with an enzymatic assay of a nucleic aciddye-based substrate (Substrate No. 19, at 10 μM) for a caspase-3 enzyme(0.1 unit/mL) in buffer, with excitation set at 485 nm and emissioncollected at 530 nm, as further described in connection with Examples 10and 53.

FIG. 4A is a graphical representation of a count, or number of cells,versus relative fluorescence associated with the detection of enzymaticactivity of a caspase-3 enzyme in live cells via flow cytometry, using acontrol of uninduced Jurkat cells (representation in FIG. 4A) as furtherdescribed in relation to Examples 10 and 54.

FIG. 4B is a graphical representation of a count, or number of cells,versus relative fluorescence associated with the detection of enzymaticactivity of a caspase-3 enzyme in live cells via flow cytometry, using anucleic acid dye-based substrate (Substrate No. 19, at 10 μM) for thecaspase-3 enzyme that was incubated for 15 minutes with Jurkat cellsthat had been induced with staurosporine (at 1 μM) for 1 hour as furtherdescribed in relation to Examples 10 and 54.

FIG. 4C is a graphical representation of a count, or number of cells,versus relative fluorescence associated with the detection of enzymaticactivity of a caspase-3 enzyme in live cells via flow cytometry, using anucleic acid dye-based substrate (Substrate No. 19, at 10 μM) for thecaspase-3 enzyme that was incubated for 15 minutes with Jurkat cellsthat had been induced with staurosporine (at 1 μM) for 2.5 hours asfurther described in relation to Examples 10 and 54.

FIG. 4D is a graphical representation of a count, or number of cells,versus relative fluorescence associated with the detection of enzymaticactivity of a caspase-3 enzyme in live cells via flow cytometry, using anucleic acid dye-based substrate (Substrate No. 19, at 10 μM) for thecaspase-3 enzyme that was incubated for 15 minutes with Jurkat cellsthat had been induced with staurosporine (at 1 μM) for 5 hours asfurther described in relation to Examples 10 and 54. FIG. 4A, FIG. 4B,FIG. 4C and FIG. 4D may be collectively referred to herein as FIG. 4.

FIG. 5A is a confocal fluorescent image of apoptotic Jurkat cells(previously induced with staurosporine) that were incubated successivelywith a substrate (Substrate No. 19, at 10 μM) for a caspase-3 enzyme for15 minutes and Texas Red-Annexin V for 15 minutes, as further describedin relation to Examples 10 and 55.

FIG. 5B is a confocal fluorescent image of apoptotic Jurkat cells(previously induced with staurosporine) that were incubated successivelywith a substrate (Substrate No. 19, at 10 mM) for a caspase-3 enzyme for15 minutes and Texas Red-Annexin V for 15 minutes, as further describedin relation to Examples 10 and 55.

FIG. 5C is a confocal fluorescent image of apoptotic Jurkat cells(previously induced with staurosporine) that were incubated successivelywith a substrate (Substrate No. 19, at 10 mM) for a caspase-3 enzyme for15 minutes and Texas Red-Annexin V for 15 minutes, as further describedin relation to Examples 10 and 55.

FIG. 5D is a confocal fluorescent image of apoptotic Jurkat cells(previously induced with staurosporine) that were incubated successivelywith a substrate (Substrate No. 19, at 10 mM) for a caspase-3 enzyme for15 minutes and Texas Red-Annexin V for 15 minutes, as further describedin relation to Examples 10 and 55.

FIG. 5E is a confocal fluorescent image of apoptotic Jurkat cells(previously induced with staurosporine) that were incubated successivelywith a substrate (Substrate No. 19, at 10 mM) for a caspase-3 enzyme for15 minutes and Texas Red-Annexin V for 15 minutes, as further describedin relation to Examples 10 and 55.

FIG. 5F is a confocal fluorescent image of apoptotic Jurkat cells(previously induced with staurosporine) that were incubated successivelywith a substrate (Substrate No. 19, at 10 mM) for a caspase-3 enzyme for15 minutes and Texas Red-Annexin V for 15 minutes, as further describedin relation to Examples 10 and 55. The cells shown in these images werefrom the same cell population. FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG.5E and FIG. 5F may be collectively referred to herein as FIG. 5.

FIG. 6 is a graphical representation of relative fluorescence emissionintensity versus dsDNA concentration (μg/mL), or a DNA titration, of anucleic acid dye-based substrate (Compound No. 21, at 1 μM in TE buffer)for a histone deacetyltransferase (HDAC) enzyme and a control compound(Compound No. 23, at 1 μM in TE buffer), as further described inconnection with Examples 23, 25 and 56.

FIG. 7 is a graphical representation of relative fluorescence emissionintensity versus liposome concentration (mM), or a liposome titration,of a membrane dye-based substrate (Compound No. 44) for a caspase-3enzyme and a control (Compound No. 46), as further described in relationto Examples 48, 50 and 57.

FIG. 8 is a schematic illustration of an interaction between an enzymesubstrate, a nucleic acid dye-based substrate for a caspase-3 enzyme,and an enzyme, the caspase-3 enzyme, that results in an enzymaticcleavage that brings about fluorescence.

FIG. 9 is a schematic illustration of an interaction between an enzymesubstrate, a membrane dye-based substrate for a caspase-3 enzyme, and anenzyme, the caspase-3 enzyme, that results in an enzymatic cleavage thatbrings about fluorescence.

FIG. 10, FIG. 11, FIG. 12, FIG. 13, FIG. 14 and FIG. 15 arepresentations of Table 2, Table 3, Table 4, Table 5 and Table 6,respectively, each of which is further described herein. FIG. 12 is usedas a collective reference herein for FIG. 12A and its sub-parts FIGS.12A-1 and FIG. 12A-2, FIG. 12B, and FIG. 12C.

FIG. 12A (including FIGS. 12A-1 and FIGS. 12A-12) contains an initialportion of Table 4.

FIG. 12B contains an intermediate portion of Table 4 and is acontinuation of FIG. 12A.

FIG. 12C contains a final portion of Table 4 and is a continuation ofFIG. 12B.

FIG. 14 is used as a collective reference herein for FIG. 14 and itssub-part FIG. 14A, sub-part FIG. 14B, and sub-part FIG. 14C.

FIG. 14A contains an initial portion of Table 6.

FIG. 14B contains an intermediate portion of Table 6 and is acontinuation of FIG. 14A.

FIG. 14C contains a final portion of Table 6 and is a continuation ofFIG. 14B.

FIG. 16A is a presentation of Panel 1.

FIG. 16B is an additional presentation of Panel 1. FIGS. 16A and 16B maybe collectively referred to herein as FIG. 16.

FIG. 17 is a presentation of Panel 2.

FIG. 18A is a presentation of Panel 3.

FIG. 18B is an additional presentation of Panel 3. FIGS. 18B and 18C maybe collectively referred to herein as FIG. 18.

FIG. 19 is a presentation of Panel 4.

FIG. 20A is a presentation of Panel 5.

FIG. 20B is an additional presentation of Panel 5.

FIG. 20C is an additional presentation of Panel 5.

FIGS. 20A, 20B, and 20C may be collectively referred to herein as FIG.20.

FIG. 21 is a presentation of Panel 6.

FIG. 22 is a presentation of Panel 7.

Each of the aforementioned Panels is further described herein.

DETAILED DESCRIPTION

In the description of the invention herein, it will be understood that aword appearing in the singular encompasses its plural counterpart, and aword appearing in the plural encompasses its singular counterpart,unless implicitly or explicitly understood or stated otherwise. Further,it will be understood that for any given component described herein, anyof the possible candidates or alternatives listed for that component,may generally be used individually or in any combination with oneanother, unless implicitly or explicitly understood or stated otherwise.Additionally, it will be understood that any list of such candidates oralternatives, is merely illustrative, not limiting, unless implicitly orexplicitly understood or stated otherwise. Still further, it will beunderstood that any figure or number or amount presented herein inconnection with the invention is approximate, and that any numericalrange includes the minimum number and the maximum number defining therange, unless implicitly or explicitly understood or stated otherwise.Additionally, it will be understood that any permissive, open, oropen-ended language encompasses any relatively permissive torestrictive, less open to closed, or less open-ended to closed-endedlanguage, respectively, unless implicitly or explicitly understood orstated otherwise. Merely by way of example, the word “comprising” mayencompass “comprising”—, “consisting essentially of”, and/or “consistingof”-type language.

Various terms are generally described below to facilitate anunderstanding of the invention. It will be understood that acorresponding general description of these various terms applies tocorresponding linguistic or grammatical variations or forms of thesevarious terms. It will also be understood that the general descriptionof any term below may not apply or may not fully apply when the term isused in a non-general or more specific manner. It will also beunderstood that the invention is not limited to the terminology usedherein, or the descriptions thereof, for the description of particularembodiments. It will further be understood that the invention is notlimited to embodiments of the invention as described herein orapplications of the invention as described herein, as such may vary.

Generally, the terms “stain” and “dye” may be used interchangeably andrefer to an aromatic molecule capable of absorbing light in the spectralrange of from about 250 nm to about 1,200 nm. Generally, the term “dye”may refer to a fluorescent dye, a non-fluorescent dye, or both.Generally, the term “fluorescent dye” refers to a dye capable ofemitting light when excited by another light of appropriate wavelength.

Generally, the term “fluorogenic” refers to a state or condition ofhaving the capability to be fluorescent following a chemical,biochemical or physical occurrence or event. Generally, the term“fluorogenic dye” refers to a non-fluorescent dye or a weaklyfluorescent dye that becomes more fluorescent (such as at least twotimes more fluorescent, for example) upon the occurrence of a chemical,biochemical, and/or physical event.

Generally, the term “fluorescence quencher” refers to a molecule capableof quenching the fluorescence of another fluorescent molecule.Fluorescence quenching can occur via at least one of the three ways. Thefirst type of fluorescence quenching occurs via fluorescence resonanceenergy transfer (FRET) (Förster, Ann. Phys. (1948); and Stryer et al.,Proc. Natl. Acad. Sci. (1967)), wherein a quencher absorbs the emissionlight from a fluorescent molecule. The absorption peak of a FRETquencher usually has to have significant overlap with the emission peakof a fluorescent dye for the FRET quencher to be an efficientfluorescent quencher. A FRET quencher is typically a non-fluorescentdye, but can also be a fluorescent dye. When a quencher is a fluorescentdye, only the absorption property of the dye is utilized. A second typeof fluorescence quenching occurs via photo-induced electron transfer(PET), wherein the quencher is an electron-rich molecule that quenchesthe fluorescence of a fluorescent molecule by transferring an electronto the electronically excited dye. A third type of fluorescencequenching occurs via dye aggregation, such as H-dimer formation, whereintwo or more dye molecules are in physical contact with one another,thereby dissipating the electronic energy into the vibrational modes ofthe molecules. This type of contact fluorescence quenching can occurbetween two identical fluorescent dyes, or between two differentfluorescent dyes, or between a fluorescent dye and a FRET quencher, orbetween a fluorescent dye and a PET quencher. Other types offluorescence quenchers, though not used as commonly, include stable freeradical compounds and certain heavy metal complexes.

Generally, the term “fluorescent nucleic acid stain” or “fluorescentnucleic acid dye” refers to a dye capable of binding to a nucleic acidto form a fluorescent dye-nucleic acid complex. A fluorescent nucleicacid dye is typically non-fluorescent or weakly fluorescent by itself,but becomes highly fluorescent upon nucleic acid binding.

Generally, the term “reactive group” may refer to a “reactive group” ora “functional group” and the term “functional group” may refer to a“reactive group” or a “functional group.” Either term may refer, or bothterms may refer, to a bond-forming group on a dye, or to a bond-forminggroup on the substrate moiety molecule. Here, by way of convenience, butnot limitation, a bond-forming group on the dye will generally bereferred to as a functional group and a bond-forming group on thesubstrate moiety molecule will generally be referred to as a reactivegroup. The reactive group and the functional group may be, and typicallyare, an electrophile and a nucleophile, respectively, that can form acovalent bond.

Generally, the term “pro-substrate” or “pro-enzyme substrate” refers toan enzyme substrate precursor that can be converted to an enzymesubstrate though a chemical, biochemical or photochemical process. Apro-substrate may be used to deliver a substrate that may be too polarto cross a cell membrane, for example. By way of example, an enzymesubstrate comprising at least one carboxylic acid group may be made intoa methyl ester or an acetoxymethyl ester (AM ester) pro-substrate, whichmay more easily enter cells. Further by way of example, an enzymesubstrate comprising at least one phosphate group may be made into apro-substrate by converting the phosphate group to a phosphate AM ester.Once in the cells, the pro-substrates may be catalytically hydrolyzedback into normal enzyme substrates.

Generally, the term “amino acid” may refer to a natural amino acid, anunnatural amino acid, a protected amino acid, an unprotected amino acid,and/or an amino acid comprising a fluorescence quencher.

Generally, the term “peptide” may refer to a peptide comprising allnatural amino acids, a peptide comprising at least one unnatural aminoacid, a peptide comprising at least one protection group, a peptidecomprising at least one ester linkage in place of a normal peptidelinkage, such as for improved enzyme kinetics, for example, and/or apeptide comprising a fluorescence quencher molecule.

Generally, when used in connection with a functional dye, the term“functionality” or “functionality strength” refers to the capacity ofthe functional dye to fluorescently bind to a partner molecule, partnermolecules, an assembly of partner molecules, and/or the like, and isgenerally defined as: functionality strength=kΦε, where k is theassociation constant between the dye and the partner molecule, partnermolecules, assembly of partner molecules, and/or the like; Φ is thefluorescence quantum yield of the dye bound to the partner molecule,partner molecules, assembly of partner molecules, and/or the like; and εis the extinction coefficient of the dye bound to the partner molecule,partner molecules, assembly of partner molecules, and/or the like.

In the design of fluorogenic enzyme substrates, one challenge concernsthe availability of suitable or desirable fluorescent dyes havingvarious suitable or desirable properties. A desirable fluorescent dyefor preparing a fluorogenic enzyme substrate should have long excitationand emission wavelengths, such as an excitation wavelength that islonger than 470 nm and emission wavelength that is longer than 500 nm.There are several dyes available, each having a different set ofexcitation/emission wavelengths to facilitate multicolor imaging whenrequired. Using long excitation and emission wavelengths in detectioncan minimize the background signal from sample containers or otherbiomolecules that may be present. The background signal may be aparticularly serious problem for detecting enzyme activity in livingcells or tissues because numerous intracellular molecules have intrinsicfluorescence in the blue fluorescence region. For imaging biologicalactivity in living animals, dyes with even longer wavelengths arerequired. Typically, near infrared (near IR) dyes with wavelengths inthe 650-1200 nm region are used because light in this wavelength regionhas better tissue penetration. (Wyatt et al., Phil. Trans. R. Soc.London B 352, 661 (1997).) For these reasons, coumarin dyes, which areusually UV-excitable and blue fluorescent dyes, are not so widely usedfor designing fluorogenic enzyme substrates. Although both rhodamine 110and fluorescein have desirable excitation and emission wavelengths inthe green color region and have been used for constructing a variety offluorogenic enzyme substrates, suitable red fluorescent dyes,particularly suitable red dyes for preparing red fluorogenic peptidasesubstrates, are lacking. A desirable dye should have only one functionalgroup to be used for covalently linking itself to an enzyme substratemoiety molecule so that only a single enzymatic cleavage is required torelease the dye, thus simplifying the kinetics of the analysis.Fluorescein and rhodamine 110, both of which have two functional groups,result in substrates that require two cleavage steps to release theparent dyes completely. A desirable dye should be minimally fluorescent,such as completely nonfluorescent, when conjugated to the substratemoiety molecule, but become highly fluorescent following the enzymaticreaction. As mentioned above, it is a challenge to find a suitable or adesirable fluorescent dye for preparing a fluorogenic enzyme substrate.

Another challenge in the design of fluorogenic enzyme substratesconcerns the use of the substrates in detecting intracellular enzymeactivity in living cells or tissues in a cell-specific manner. Someenzyme assays, such as ELISA and some of the preliminary drugscreenings, are carried out in cell-free systems with an isolatedenzyme. Some enzyme assays may be carried out with cell lysates. In someapplications, however, detecting enzyme activity in living cells ishighly desirable or necessary. For example, the ability to fluorescentlydetect a reporter enzyme in living cells in a cell-specific mannerpermits one to study gene expression by flow cytometry and microscopy. Asubstrate comprising a near IR dye may be used to image intracellularenzyme activity in live animals in real-time. Further by way of example,intracellular enzyme detection may be used for high-throughput drugscreening. Although screening drug candidates with an isolatedintracellular enzyme in a cell-free system can yield useful information,it does not account for the fact that many intracellular enzymes exerttheir activities in concert with other cellular receptors and cofactors,and does not test for the potential toxicity of the drug candidates andtheir efficiency in crossing cell membranes. Cell-based drug screeningoffers far more pharmacologically relevant information concerning notonly the interaction between the drug candidate and the target bindingmolecule, but also the interaction between the drug candidate and theentire cellular environment.

In the design of fluorogenic enzyme substrates for intracellular enzymedetection, two issues should be addressed. One issue concerns themembrane permeability of the substrate, which is determined primarily bythe individual membrane permeability of the substrate moiety moleculeand the dye. Since only limited chemical modification of the substratemoiety molecule may be employed to improve its membrane permeabilitywithout changing its enzyme kinetics, it is useful to select a dye thathas good membrane permeability. Another issue concerns the retention ofthe fluorescent product within the cell following the enzymaticreaction. One of two approaches is generally employed to facilitate suchretention. One approach is to prepare an enzyme substrate using a dyewith a reactive group, such as a haloalkyl group (U.S. Pat. No.5,576,424) or a perfluorobenzoyl thiol-reactive group (Zhang et al.,Bioconjugate Chem. 14, 458 (2003)), that is capable of conjugating thedye or the enzyme substrate to the highly polar glutathione or proteinswithin the cells under physiological condition. Once the enzymesubstrate is hydrolyzed, the resulting dye-glutathione or dye-proteinconjugate is trapped within the cells by virtue of high polarity, thusproducing a fluorescence signal specific to the cells having the enzymeactivity. Another approach is to prepare an enzyme substrate using a dyewith a lipophilic tail, which both facilitates the delivery of thesubstrate moiety into the cytoplasm and enables the enzymaticallyreleased dye to remain in the cytoplasmic membranes, resulting in acell-specific fluorescent signal. (U.S. Pat. No. 5,208,148; Cai et al.,Bioorg. Med. Chem. Lett. 11, 39 (2001)).

A fluorogenic enzyme substrate of the present invention comprises anenzyme substrate moiety molecule and a biological functional dye. Theenzyme substrate comprises dual functionality, being able to detectenzyme activity and to fluorescently stain another biomolecule and/orother biomolecules, such as an assembly of biomolecules, for example.The enzyme substrate may be useful in the detection of intracellularenzyme activity in a living cell because the enzymatically released dyemay not only remain within the cell, but may also offer information onthe quantity, distribution, morphology of a component of the cell,and/or the like, by way of example. The enzyme substrate may be usefulfor a variety of applications, such as the study of gene expression,drug screening, disease diagnosis, and/or the like, by way of example.At least some enzyme substrates of the invention comprise a nearinfrared (near IR) functional dye, such that the enzyme substrate may besuitable for imaging enzyme activity in real-time in living animals.

A fluorogenic enzyme substrate of the present invention may be describedby the general Formula 1 set forth below, or biologically acceptablesalts or pro-enzyme substrates thereof.DYE-(B)_(m)  Formula 1In Formula 1, the DYE by itself is a biologically functional fluorescentor fluorogenic dye capable of binding to a partner biomolecule and/orpartner biomolecules, such as an assembly of partner biomolecules;independently, each B is an enzyme substrate moiety molecule capable ofbeing transformed by an enzyme; and the subscript, m, indicates a numberof substrate moiety molecule(s). The subscript, m, may be 1, 2, 3, 4 or5. The enzymatic substrate transformation may comprise any of a varietyof enzymatic actions, such as bond cleavage between the DYE and each B,bond cleavage within each substrate moiety molecule B, and or bondformation associated with each B, such as phosphorylation of each B, forexample.

Herein, a biologically functional fluorogenic or fluorescent dyegenerally refers to a biological stain, or a dye that impartsfluorescence upon physical association with a partner biomolecule orwith other partner biomolecules, such as an assembly of partnerbiomolecules. A partner biomolecule may be any of a variety of suitablebiomolecules, such as DNA, RNA, a protein, and/or a biological receptor,merely by way of example. An assembly of partner biomolecules may be anyof a variety of suitable assemblies, such as an assembly in the form ofa protein assembly, such as an actin filament and/or a microtubule,merely by way of example, cell organelles, a cell organelle membrane,and/or a cytoplasmic membrane, merely by way of example. The fluorogenicor fluorescent functional dye may be any of a variety of suitable suchdyes, such as a DNA dye, a RNA dye, a fluorescent ligand for a protein,proteins, a protein assembly, and/or protein assemblies, merely by wayof example, a cytoplasmic membrane dye, a cell organelle dye, such as adye for a mitochondrion, a Golgi body, an endoplasmic reticulum, alysosome, and/or an endosome, merely by way of example. Further by wayof example, as to fluorescent ligand dyes, fluorescent ligands, which,generally speaking, are fluorescently-labeled small organic moleculesthat may bind to a protein-based receptor molecules, may be used for theintracellular mapping of target protein molecules. For example,fluorescently-labeled taxols (J. Biol. Chem. 275, 26265 (2000)) may beused to visualize microtubules in cells and fluorescently-labeledphalloidins may be used to map actin filaments in cells (J. Biol. Chem.273, 11144 (1998). According to an embodiment of the invention, the DYEcomprises a fluorogenic functional dye. The fluorogenic functional dyemay comprise a nucleic acid dye or an organelle dye, such as amitochondrial dye, a vacuole dye, an endoplasmic reticulum (ER) dye,and/or a lysosomal dye, merely by way of example.

In Formula 1, the enzyme substrate moiety molecule B is the portion ofthe fluorogenic or fluorescent enzyme substrate that interacts directlywith an enzyme. Generally speaking, the molecule B may resemble thenatural substrate of the enzyme. The enzyme substrate transformationdescribed above may involve a catalytic bond cleavage between the DYEand B to produce a fluorogenic or fluorescent product DYE; a catalyticbond cleavage within B to produce a fluorogenic or fluorescent productDYE-(B′)_(m), where B′ comprises a portion of B and m is 1, 2, 3, 4 or5; or a catalytic bond formation on B to produce a fluorogenic orfluorescent product DYE-(B″)_(m), where B″ comprises B with at least oneadditional chemical group and m is 1, 2, 3, 4 or 5. A catalytic bondcleavage may be any suitable such cleavage, such as hydrolysis of apeptide catalyzed by a peptidase, hydrolysis of an ester catalyzed by anesterase, hydrolysis of a backbone phosphate of DNA or RNA catalyzed bya nuclease, dealkylation of an aromatic ether catalyzed by a dealkylaseor cytochrome P450, deacetylation of acylated lysine side chains inhistone or a similar peptide catalyzed by histone deacetylase (HDAC), orthe like, merely by way of example. A catalytic bond formation may beany suitable such formation, such as phosphorylation of a peptide or acarbohydrate substrate moiety catalyzed by a kinase, acylation of alysine residue side chain in a histone-mimicking peptide catalyzed byhistone acetyltransferase (HAT), alkylation of glutathione thiolcatalyzed by glutathione transferase, or the like, merely by way ofexample.

The enzyme substrate moiety molecule B may be any of a variety suitablesuch molecules, such as an amino acid that is used with a peptidase, apeptide that is used with a peptidase, an α-amino-protectedε-N-acetyllysine that is used with a histone deacetyltransferase (HDAC),a phosphoryl that is used with an alkaline or acid phosphatase, asulfuryl that is used with a sulfatase, a carbonyl that is used with anesterase, an alkyl that is used with a cytochrome P450 enzyme, aglycosidyl that is used with a glycosidase, a phosphorylatedphosphatidylinositol or an unphosphorylated phosphatidylinositol that isused with a phosphatidylinositol-specific phospholipase C, aglycosylated phosphatidylinositol that is used with aphosphatidylinositol-specific phospholipase C, an adenosine-5′-phosphatethat is used with a phosphodiesterase, a nucleoside-3′-phosphate that isused with a nuclease or a ribonucleaseptide, and/or the like, merely byway of example.

When the enzyme substrate moiety molecule B is a peptide or amino acid,the peptide or the amino acid may be linked to DYE in a number of ways.One way, referred to herein as “C-linkage,” comprises the linkage of theC-terminal carboxylic group of a peptide or the α-carboxylic group of anamino acid to an amine group on the DYE to form a peptide bond, whichmay or may not be the scissile bond. Enzymatic cleavage of the substrateprepared via C-linkage results in a product DYE that comprises apositively charged amine. Another way, referred to herein as“N-linkage,” comprises the linkage of the N-terminal amine group of apeptide or the α-amine group of an amino acid to a carboxylic group onthe DYE to form a peptide bond, which may or may not be the scissilebond. Enzymatic cleavage of the substrate prepared via N-linkage resultsin a product DYE that comprises a negatively charged carboxylate group.The choice of using C-linkage or N-linkage to produce a peptidasesubstrate may be based on the enzymatic cleavage requirement of thepeptide/peptidase pair, whether the cleavage pattern will enhance thefunctionality of the functional dye following the enzymatic reaction,and/or the like, merely by way of example. Generally speaking, such achoice may be made in favor of the linkage that results in a substratewith low functionality and a fluorogenic or fluorescent product withhigh functionality, as use of such a substrate may provide a positivedetectable signal, which may be advantageous relative to a negativedetectable signal. Generally speaking, such a choice may be facilitatedby reference to information on the sequence of a peptide substratemoiety, the relationship between the structure and the functionality ofa functional dye, and/or the like, merely by way of example.

According to an embodiment of the invention, the substrate moietymolecule B is a peptide or an amino acid that comprises a fluorescencequencher attached to the portion of B that is cleaved from the dyefollowing the enzymatic transformation of the substrate. A substrate ofsuch construction may have improved signal-to-noise ratio, as anybackground fluorescence due to the interaction between the substrate andnucleic acid may be significantly reduced by FRET effect.

According to an embodiment of the invention, the fluorogenic orfluorescent substrate comprises only one substrate moiety B. Accordingto another embodiment, the fluorogenic or fluorescent substratecomprises two substrate moiety molecules B, wherein each B may be thesubstrate moiety molecule for the same enzyme, or one B may be thesubstrate moiety molecule for an enzyme and the other B may be thesubstrate moiety molecule for another enzyme. According to an embodimentof the invention, each of the two substrate moiety molecules B is thesubstrate moiety for the same enzyme.

An enzyme substrate of the invention may produce a detectablefluorescence signal in response to enzyme activity by way of a processthat comprises enzymatically transforming the enzyme substrate toproduce a fluorogenic or fluorescent product DYE, DYE-(B′)_(m) orDYE-(B″)_(m), and allowing the fluorogenic or fluorescent product and apartner biomolecule, partner biomolecules, and/or an assembly of partnerbiomolecules to interact or to bind. A detectable fluorescence signalmay comprise a positive signal or a negative signal, as may depend onthe relative functionality strengths of the enzyme substrate and thecorresponding fluorogenic or fluorescent product of the enzymaticreaction. For example, a positive signal may result if the fluorogenicor fluorescent enzymatic product has a stronger functionality than thestarting enzyme substrate. Further by way of example, a negative signalmay result if the fluorogenic or fluorescent enzymatic product has aweaker functionality than the starting enzyme substrate. In a case inwhich the substrate is applied to a living cell or living cells, adetectable fluorescence signal may comprise a change in the fluorescentstaining pattern of the cell or cells, a combination of changes in thedetected fluorescence intensity and fluorescent staining pattern of thecell or cells, and/or the like, merely by way of example.

According to various embodiments of the invention, enzymatictransformation of an enzyme substrate produces a positive fluorescencesignal. For example, in one such embodiment, enzymatic transformation ofthe starting enzyme substrate, DYE-(B)_(m) of Formula 1, comprises ahydrolytic cleavage of the bond between the DYE and B, which results ina fluorogenic product that is substantially more functional (such as atleast two times more functional, for example) than the starting enzymesubstrate, thus producing a positive detectable signal. Further by wayof example, in another such embodiment, enzymatic transformation of thestarting enzyme substrate, DYE-(B)_(m) of Formula 1, comprises ahydrolytic cleavage of a bond within substrate moiety B to generate afluorogenic product DYE-B′, wherein B′ is a portion of B that remainsattached to the DYE, that is substantially more functional (such as atleast two times more functional, for example) than the startingsubstrate, thus producing a positive detectable signal.

The sensitivity of enzyme detection associated with the use of an enzymesubstrate DYE-(B)_(m) may be at least partly determined by thebackground signal, as may be determined by the functionality strength ofthe enzyme substrate. Generally, a DYE-(B)_(m) of weaker functionalitycorresponds to a weaker interaction between the enzyme substrate and apartner biomolecule, partner biomolecules, and/or an assembly of partnerbiomolecules, and thus, a weaker background fluorescence signal.Accordingly to an embodiment of the invention, the fluorogenic enzymesubstrate DYE-(B)_(m) may have minimal or no functionality.

Any of various suitable methods, or any suitable combination of methods,by which a substrate moiety molecule B conjugated to the DYE reduces oreliminates the functionality of the DYE, may be employed. According toone method, a substrate moiety molecule B may serve as a steric blockthat is strategically attached to the DYE such that it interferes withthe functionality of the DYE. Such a method may be suitable when the DYEis a nucleic acid dye or a biological ligand for a receptor, wherein thenucleic acid dye or the biological ligand may have to possess or toassume a shape that is compatible with the binding site on the nucleicacid or the receptor.

According to another method, a substrate moiety molecule B may carry anet positive charge or a net negative charge so that upon conjugation tothe DYE, the substrate moiety molecule B alters the amount and/or thenature of the charge on the DYE that is associated with thefunctionality of the DYE. Such a method may be suitable for a DYE whosefunctionality is sensitive to the amount and/or the nature of the chargeit carries. For example, most nucleic acid dyes are positively charged.Generally speaking, additional positive charge or charges may enhancethe nucleic acid binding associated with a nucleic acid dye. (U.S. Pat.No. 5,321,13.) Attaching a substrate moiety molecule B that bears one ormore negative charge(s) to the DYE may be expected to weaken or tocompletely diminish the nucleic acid binding ability of the DYE. (SeeFIGS. 1-6 and 8, and associated Examples, for example.) Further by wayof example, mitochondrial dyes generally bear a delocalized positivecharge necessary for the dyes to partition into mitochondrial membranes,where there is usually a large negative membrane potential that attractspositively charged dyes. (Biotium, Inc., Fluorescent Probes and RelatedBiochemical Reagents for Life Science, 2005-2006, and references setforth therein.) Attaching a negatively charged substrate moiety moleculeB to a mitochondrion-staining DYE may render the DYE less functional ornonfunctional. (See Examples 33 and 39, for example.)

According to yet another method, a lipophilic functional dye DYE may bemade less functional or nonfunctional by the attachment of a substratemoiety molecule B having high hydrophilicity. According to yet anothermethod, a hydrophilic functional dye DYE may be made less functional ornonfunctional by the attachment of a substrate moiety molecule B havinghigh hydrophobicity. For example, a fluorogenic membrane DYE may be madeless functional or nonfunctional via conjugation to a highlywater-soluble substrate moiety molecule B. The resulting enzymesubstrate, having increased water solubility by virtue of thisconjugation, has a reduced ability or no ability to partition intomembranes and fluoresce. (See FIGS. 7 and 9, and associated Examples,for example.) Further by way of example, a DNA-binding dye DYE may bemade less functional or nonfunctional by conjugation to a membrane-boundsubstrate moiety molecule B, such as a membrane-bound hydrophobicpeptide, for example. The resulting enzyme substrate, residing more orfully in the cell membranes, has a reduced ability or no ability tointeract with the DNA in the cell nucleus until a membrane-bound enzymecleaves the enzyme substrate to release the DYE. (See Substrate No. 36of Table 4.)

The enzyme substrates may be prepared via a process that is nowgenerally described. The process may comprise appreciating, identifyingor determining any of various suitable properties or qualities of thesubstrate moiety molecule B relative to a given enzyme, such asstructural configuration, bulkiness, the amount of any charge, thenature of any charge, hydrophilicity, hydrophobicity, and/or the like,merely by way of example. The process may comprise selecting afunctional dye that may be reduced in functionality or de-functionalizedby the substrate moiety molecule B. Such a selection may be based on anyof the properties or qualities of the substrate moiety molecule B, orany other useful criteria. The process may comprise derivatizing thefunctional DYE so that it has a suitable functional group forconjugating the substrate moiety molecule B to the DYE. The processcomprises conjugating the DYE to the substrate moiety molecule B or aprecursor of the substrate moiety molecule B. The process may compriseconverting the conjugate to the enzyme substrate.

Once the enzyme substrate is prepared, it may be tested. For example,the enzyme substrate may be incubated with an enzyme for which theenzyme substrate was selected or designed in the presence of at leastone partner biomolecule, and/or at least one assembly of partnerbiomolecules, as may be appropriate in connection with a cell-freeapplication, while the enzymatic reaction is monitored using a suitabledetection device, such as a fluorescence microplate reader or afluorometer, for example. Any detectable change in fluorescenceintensity may be taken as an indicator that the substrate may besuccessfully used for an intended enzyme detection application in acell-free system. (See Example 53, for example.) Further by way ofexample, the enzyme substrate may be incubated in at least one cellculture for an appropriate amount of time, such as about 5 minutes ormore, wherein the cells of the cell culture contain an enzyme for whichthe enzyme substrate was selected or designed, as may be appropriate inconnection with a cellular application, while the cell culture ismonitored for enzymatic activity using a suitable detection device, suchas a fluorescence microscope, a fluorescence flow cytometer, or afluorescence microplate reader, for example. Any detectable change influorescence intensity and/or fluorescence staining pattern within thecells may be taken as an indicator that the substrate may besuccessfully used for an intended enzyme detection application in acellular system. Further, once the enzyme substrate is prepared, it maybe modified. For example, the enzyme substrate may be modified tofacilitate obtaining a better or an optimized signal, signal-to-noiseratio, and/or the like, associated with enzyme activity detection. Sucha modification may comprise modifying the substrate structure, forexample, by varying the position at which the substrate moiety moleculeB is attached to the DYE, varying the length of the linker between thesubstrate moiety molecule B and the DYE, attaching an additionalfluorescence quencher molecule to the substrate moiety molecule B,and/or the like, merely by way of example.

Enzyme substrates of the present invention may vary according to thetype of functional dyes that are used to produce them. Methods forpracticing various aspects of the invention may also vary accordingly.Merely by way of example, various enzyme substrates and associatedmethods are provided below.

Enzyme Substrate Comprising Nucleic Acid Dye

An enzyme substrate of the present invention may comprise a nucleic aciddye. For the most part, a nucleic acid dye is either an intercalatingdye (intercalator) or a minor-groove binding dye (minor groove binder).An intercalator is a dye that inserts itself in between two neighboringbase pairs of double-stranded DNA. A minor groove binder is a dye thatbinds to the minor groove of double-stranded DNA.

The amount of charge on a nucleic acid dye may affect, perhapsprofoundly so, the nucleic acid binding ability of the dye. Addingpositively charged groups to an intercalator or a minor groove bindermay enhance the nucleic acid binding of the nucleic acid dye as a resultof the increased electrostatic attraction between the positively chargeddye and the negatively charged DNA. (Gaugain et al., Biochemistry 17,5071 (1978); U.S. Pat. No. 5,321,130.) In general, a nucleic acid dyewith high nucleic acid binding affinity may provide a high fluorescencesignal in the presence of nucleic acid, as a high percentage of the dyemay be in the nucleic acid-bound form, which is the fluorescent form ofthe dye. Attaching negatively charged groups to a nucleic acid dye maydecrease or completely diminish the nucleic acid binding ability of thedye, as there may be repulsive interaction between the negativelycharged DNA and negatively charged dye.

The functionality of a nucleic acid dye may be sensitive to changesother than changes in the amount and/or the nature of the chargeassociated with the dye. By way of example, any of various structuralmodifications of the nucleic acid dye may affect its functionality.Further by way of example, an intercalator or a minor groove bindingnucleic acid dye, may have to have or to assume a shape that fits abinding site of a nucleic acid in order for intercalation or minorgroove binding to occur. Attaching a sterically bulky group to thenucleic acid dye at a suitable position may disrupt the nucleic acid-dyeinteraction, rendering the dye weakly functional or completelynonfunctional. The relationship between the structure and thefunctionality of a nucleic acid dye may be exploited in the design ofenzyme substrates of the invention, as may be illustrated in thedescription below.

A negatively charged substrate moiety molecule B may be covalentlylinked to a nucleic acid dye to form an enzyme substrate. The nucleicacid dye of the enzyme substrate may have reduced functionality or maybe substantially de-functionalized relative to the original nucleic aciddye by virtue of the negative charge associated with the originalsubstrate moiety molecule B, and/or by virtue of steric bulkiness. In anenzymatic cleavage reaction, B or a portion of B associated with thenegative charge may be removed to generate a fluorogenic product DYE orDYE-B′, which is substantially functional (such as at least two timesmore functional than the dye of the starting substrate, for example) asa nucleic acid dye. A positive signal may then be generated upon thebinding of DYE or DYE-B′ to nucleic acid.

A suitable substrate moiety molecule B may be selected according to itsapplication, such as its use in connection with a suitable enzyme.Merely by way of example, a negatively charged amino acid or peptidylmay be used in an application involving a peptidase (see FIGS. 1-5 and8, and associated Examples, for example), a suitable substrate moietymolecule may be used in an application involving a lactamase (seeExample 45, for example), a phosphoryl may be used in an applicationinvolving a phosphatase, a negatively charged glycosidyl may be used inan application involving a glycosidase (see Substrate No. 38 of Table 4,for example), a sulfuryl may be used in an application involving asulfatase, a phosphatidylinosityl may be used in an applicationinvolving a phosphatidylinositol-specific phospholipase C (see SubstrateNos. 39 and 40 of Table 4, for example), and/or the like. Further by wayof example, a peptide or an amino acid may be linked to a functional dyevia a C-linkage or a N-linkage, as previously described. A C-linkage maybe used to link the substrate moiety molecule B to the DYE, so that apositively charged amine may be created on the DYE following theenzymatic reaction. A DYE of increased positive charge may have enhancedfunctionality, which may result in a positive detectable signal, aspreviously described.

A neutral substrate moiety molecule B may be covalently linked to anucleic acid dye at a strategic position of the dye to form an enzymesubstrate of the invention. Such a substrate moiety may disrupt theinteraction between the dye of the enzyme substrate and nucleic acid,until the substrate moiety is sufficiently removed by the enzymaticaction to form a more functional nucleic acid dye that may provide apositive detectable signal. Such a neutral substrate moiety molecule Bmay be any of various suitable such molecules, such as a peptide thatmay be used in an application involving an elastase (see Example 26, forexample), an ε-N-acetyllysine residue that may be used in an applicationinvolving histone deacetyltransferase (HDAC) (see Example 23, forexample), neutral glycosidyls that may be used in an applicationinvolving a glycosidase, an alkyl that may be used in an applicationinvolving cytochrome P450, and/or the like, merely by way of example.

A lipophilic substrate moiety molecule B may be covalently linked to anucleic acid dye to form an enzyme substrate of the invention, such asone that may reside in a cell membrane, for example. The membrane-boundenzyme substrate may be catalytically cleaved by a membrane enzyme torelease a functional nucleic acid dye, DYE, which may then migrate tothe cell nucleus to stain nuclear DNA. (See Substrate No. 36 of Table 4,for example.) Merely by way of example, a cytoplasmic membrane-boundprotease may be γ-secretase, an enzyme associated with the formation ofβ-amyloid plaque associated with Alzheimer's disease.

A highly functional fluorogenic or fluorescent enzyme substrate may beprepared by attaching a positively charged substrate moiety molecule Bto a nucleic acid dye. Enzymatic cleavage of the substrate may provide aproduct DYE or DYE-B′, where B′ is a portion of B that does not carry apositive charge, that is less functional than the dye of the enzymesubstrate. In such a case, relative to the interaction between theenzyme substrate and nucleic acid and the associated fluorescencesignal, the interaction between the product DYE or DYE-B′ and nucleicacid and the associated fluorescence signal, respectively, are weaker,and the detected fluorescence signal is negative. Merely by way ofexample, an enzyme substrate may comprise a nucleic acid dye and apolycationically charged peptide substrate moiety molecule B, and anenzymatic reaction may remove B or a portion of B that carries positivecharge, such that the product DYE or DYE-B′ is a less functional nucleicacid dye.

A functional fluorogenic or fluorescent substrate may be prepared from anucleic acid dye covalently attached to a substrate for a kinase. Uponenzymatic phosphorylation of the substrate by the kinase, thefunctionality of the substrate may be weakened, such that a negativesignal is produced.

Virtually any nucleic acid dye may be used as the DYE in the preparationof DYE-(B)_(m) according to any of the methods described above, or anycombination of such methods, provided that the nucleic acid dye has asuitable functional group or the nucleic acid dye may be derivatized tohave a suitable functional group. Herein, DYE may refer to a nucleicacid dye that already comprises a functional group or a nucleic acid dyethat is derivatized to comprise a functional group. A functional groupmay react with a reactive group to form a covalent bond. Herein, anucleic acid dye comprising a suitable functional group may becovalently attached to a suitable substrate moiety or a suitableprecursor substrate moiety comprising a suitable reactive group.

An example of a suitable nucleic acid dye may be used as in thepreparation of DYE-(B)_(m) may be any of the intercalating3,8-diamino-6-phenylphenanthrodium dyes. The two aromatic amino groupsof such a 3,8-diamino-6-phenylphenanthrodium dye are associated with theunique spectral property and the nucleic acid binding property of thedye. Any chemical modification of the amino groups may affect, perhapssignificantly so, such a property or such properties of the dye. Theseamino groups may serve as ideal sites for attaching a substrate moietymolecule B so that a positive detectable fluorescence signal may begenerated following the cleavage of the substrate moiety molecule B fromthe dye. A general representative structure for an enzyme substrateprepared using a 3,8-diamino-6-phenylphenanthrodium and a one- ortwo-peptide substrate moiety molecule B is shown as Structure 1 below.

According to an embodiment of the invention, an enzyme substrateassociated with Structure 1 may be generally described as follows. Thesubstrate moiety molecule B may be a peptide or an amino acid. At leastone of R₁ and R₂ may be B, where the C-terminal carbonyl of B forms theenzymatically cleavable peptide bond with the amino group of the dye.When R₁ or R₂ is not B, it is H.

R₃ may be a substituted or an unsubstituted C1 to C10 alkyl. The size ofR₃ may affect the nucleic acid-binding affinity of the dye, as well asthe membrane permeability of the corresponding enzyme substrateDYE-(B)_(m). When B is a charged peptide, R₃ may be a C4 to a C10 alkyl,or a C4 to a C8 alkyl. When B is a neutral peptide, R₃ may be a C1 to aC6 alkyl.

The nucleic acid-binding affinity of the dye generally decreases as thesize of R₃ increases. When the enzyme substrate is employed to detectenzyme activity in a cell-free system, R₃ may be a small alkyl,typically a methyl or an ethyl. When the enzyme substrate is employed todetect enzyme activity in live cells, DYE-(B)_(m) should be ofsufficient membrane-permeability, which is generally proportional to theoverall hydrophobicity of the substrate. A larger R₃ may help enhancethe hydrophobicity and thus the membrane-permeability of the substrate,but may compromise the overall performance of the substrate by loweringthe nucleic acid binding affinity of the dye. An optimal range for thesize of R₃ may be one that provides an enzyme substrate of Structure 1comprising a suitable membrane-permeability for the substrate, asuitable nucleic acid binding affinity for the dye, or both. The choiceof a suitable R₃ may also depend on the nature of the substrate moietymolecule B, which itself may either increase or decrease thehydrophobicity of the substrate.

ψ may be a positively charged or negatively charged, biologicallycompatible counter ion. ψ may serve to balance the overall charge of theenzyme substrate, such that the enzyme substrate is neutral in overallcharge. Merely by way of example, a positively charged ψ may be H⁺, Na⁺,K⁺, NH₄ ⁺, N,N,N-triethylammonium, N,N-diisopropylethylammonium, or thelike. Merely by way of example, a negatively charged ψ may be a halide,a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, atetrafluoroborate, or the like. d is a number of ψ that serves tobalance the charge of the enzyme substrate.

The 8-amino group of the dye is generally more reactive than the 3-aminogroup of the dye. Thus, generally, the predominant product ofconjugation of B to the dye is a substrate having a single B attached tothe 8-amino group, in relatively high yield and of relatively simpleenzyme kinetics.

Merely by way of example, a list of enzyme substrates that are generallyrepresented by Structure 1 above is provided in Table 2 of FIG. 10,along with information concerning same. Some general conventions applyto Table 2, as now described. In the columns labeled R₁ and R₂ of Table2, bold-faced type is used to indicate amino acid residues associatedwith enzyme recognition and normal-faced type is used to indicateprotection groups for amino acids or peptides. Additionally, in thecolumn labeled R₁, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 areassociated with certain of the amino acid residues shown in bold-facedtype. In the column labeled “Cellular Localization Site of EnzymeSubstrate,” an indication of the approximate cellular location of theenzyme substrate, if the enzyme substrate is applied to living cells, isprovided. In this column, “membrane-impermeable” is indicated when theenzyme substrate may not be able to cross a cell membrane forintracellular enzyme activity detection. Such membrane-impermeablesubstrates may be used for cell-free enzyme activity detection, such asin a cell-free system of cell lysates, for example, or may be usedextracellular enzyme activity detection, such as in a live cell system,for example. In general, each of the enzyme substrates may be used in acell-free system, regardless of its cell membrane-permeability. In thecolumn labeled “Cellular Site of DYE,” an indication of the binding siteof DYE following enzymatic cleavage is provided. Generally, in aliving-cell system, the enzyme substrate is enzymatically cleaved togenerate DYE at a site where the enzyme substrate is initially located,and the resulting DYE migrates to the cell nucleus where the DYE bindsto dsDNA. Depending on the binding selectivity of the DYE to DNA andRNA, some of the DYE may bind to RNA in the cytoplasm. Finally, in thecolumn labeled “λ_(abs)/λ_(em) (nm/nm),” the wavelengths provided referto the wavelengths for the DNA-bound DYE.

According to an embodiment of the invention, the substrate moietymolecule B may be attached via a linker L to the 5-position of a3,8-diamino-6-phenylphenanthrodium. A general representative structurefor an enzyme substrate prepared using such a substrate moiety moleculeB is shown as Structure 2 below.

In the enzyme substrate generally represented by Structure 2 above, Lmay be an aliphatic C2 to about C10 linker, optionally comprising anaryl and at least one hetero atom, such as any heteroatom of theheteroatoms O, S, N, F, and Cl, for example. L may be a relatively shortlinker, such as a C2 to a C6 linker. In such a case, the substratemoiety molecule B may affect the functionality of the dye to a greaterextent. W may be an atom or atoms suitable for the formation of ascissile bond between the DYE and B for a specific enzyme, or may be anatom or atoms suitable for the formation of a covalent link between Band the DYE, wherein the enzymatic bond formation site is on B or theenzymatic cleavage site is within B. W may be any atom or atoms of atomsO, NH and S. The substrate moiety molecule B may be an amino acid or apeptide attached to W via a C-linkage, such as an amino acid that isused with a peptidase, or a peptide that is used with a peptidase, eachof which may be referred to as a peptidase substrate moiety; anα-amino-protected ε-N-acetyllysine that is used with a histonedeacetyltransferase (HDAC), which may be referred to as a HDAC substratemoiety; a phosphoryl that is used with an alkaline or an acidphosphatase, which may be referred to as a phosphatase substrate moiety;a sulfuryl that is used with a sulfatase, which may be referred to as asulfatase substrate moiety; a carbonyl that is used with an esterase,which may be referred to as an esterase substrate moiety; an alkyl thatis used with a cytochrome P450 enzyme, which may be referred to as acytochrome P450 substrate moiety; a β-D-glucuronidyl that is used with aβ-glucuronidase, a β-D-galactopyranosidyl that is used with aβ-D-galactosidase, an α-D-galactopyranosidyl that is used with anα-D-galactosidase, an α-D-mannopyranosidyl that is used with anα-D-mannosidase, an α-D-glucopyranosidyl that is used with anα-D-glucosidase, a β-D-glucopyranosidyl that is used with aβ-D-glucosidase, a X-β-D-cellobiosidyl that is used with aβ-cellobiosidase, a N-acetyl-β-D-galactosaminidyl that is used with aneurominidase, a N-acetyl-β-D-glucosaminidyl that is used with aN-acetyl-β-D-glucosaminidase or a chitinase, each of which is aglycoside and may be referred to as a glycosidase substrate moiety; aphosphorylated or an unphosphorylated phosphatidylinositol that is usedwith a phosphatidylinositol-specific phospholipase C, or a glycosylatedphosphatidylinositol that is used with a phosphatidylinositol-specificphospholipase C, each of which may be referred to as aphosphatidylinositol-specific phospholipase C substrate moiety; anadenosine-5′-phosphate that is used with a phosphodiesterase, which maybe referred to as a phosphodiesterase substrate moiety; anucleoside-3′-phosphate that is used with a nuclease or a ribonuclease,which may be referred to as a ribonuclease substrate moiety; a substratemoiety that is used with a β-lactamase, which may be referred to as aβ-lactamase substrate moiety; or the like, merely by way of example. InStructure 2, d and ψ are as previously described in connection withStructure 1.

According to an embodiment of the invention, the substrate moietymolecule B may be a peptide or amino acid that comprises at least oneenzymatically removable negative charge (ERNC). Merely by way ofexample, a peptide substrate moiety B may have at least two ERNCs. AnERNC generally refers to a negative charge from an aspartic acidresidue, a glutamic acid residue, or a negatively charged modifyinggroup that is attached to the peptide or the amino acid that is removedfrom the nucleic acid dye as a result of the enzymatic cleavage of thesubstrate moiety molecule B. A nucleic acid dye-based enzyme substratethat carries more ERNCs may generally be associated with a greaterfluorescence signal-to-noise ratio. An excessive amount of negativecharge may render it more difficult for the enzyme substrate to crosscell membranes. Thus, a consideration of how much negative charge issuitable may require some balance. Merely by way of example, inintracellular enzyme detection, the number of negative charges on anenzyme substrate may be less than 5, or less than 4. Merely by way ofexample, in other applications, such as cell-free enzyme detection orextracellular enzyme detection in a cell culture, tissue sample, or in aliving animal, for example, generally speaking, there may be no limit onthe number of ERNCs a nucleic acid dye-based enzyme substrate may carry.The charge of a neutral peptide substrate moiety or a peptide substratemoiety that has only one ERNC may be made more negative withoutaffecting the specificity of the enzyme substrate for the enzyme, as maybe accomplished by covalently attaching a negatively charged or apoly-negatively charged modifying group to the N-terminal of thesubstrate moiety. By way of example, a single negative charge may beadded to a neutral peptide via attachment of a glutamic acid thereto, orvia reaction of the N-terminal α-amine of the neutral peptide andsuccinic anhydride. Further by way of example, a triple negative chargemay be added to the neutral peptide via attachment of a tri-asparticpeptide to the N-terminal of the neutral peptide.

According to an embodiment of the invention, the substrate moietymolecule B is a peptide or an amino acid that comprises a fluorescencequencher attached to the portion of B that is cleaved from the dyefollowing the enzymatic transformation of the substrate. Merely by wayof example, the substrate moiety molecule B may comprise a peptide thatcomprises a fluorescence quencher, wherein the fluorescence quencher isattached to the N-terminal of an enzyme substrate moiety for an enzyme,such as a caspase enzyme, for example, and wherein the fluorescencequencher is sufficient for removal from the dye upon enzymatictransformation of the substrate.

According to an embodiment of the invention, the substrate moietymolecule B may be a neutral substrate moiety that reduces orsubstantially eliminates the functionality of the nucleic acid dye.Merely by way of example, the substrate moiety molecule B may act as asteric impediment or block that interferes with the nucleic acid bindingof the dye.

According to an embodiment of the invention, the substrate moietymolecule B may be a substrate moiety for β-lactamase. Merely by way ofexample, general representative structures for two such substratemoieties are shown as Sub-structure 1 and Sub-structure 2, respectively,below.

In either of Sub-structure 1 and Sub-structure 2, the dotted linerepresents the covalent bond between the substrate moiety and W shown inStructure 2. A may be a substituted C1 to C20 alkyl or aryl, optionallycomprising at least one hetero atom, such as N, O and/or S, for example,and optionally comprising at least one negatively charged group, such asa carboxylate, a sulfonate, and/or a phosphate, for example; or A may bea fluorescence quencher; and c may be 0, 1 or 2. Examples of substratemoieties for β-lactamase appear in U.S. Pat. Nos. 5,741,657, 5,955,604,and 6,031,094, and U.S. Patent Application Publication Nos. 2005/0227309and 2005/0118669. Any such substrate moiety may be combined with anucleic acid dye to prepare a fluorescent or fluorogenic β-lactamasesubstrate.

Merely by way of example, a list of enzyme substrates that are generallyrepresented by Structure 2 above is provided in Table 3 of FIG. 11,along with information concerning same. Some general conventions applyto Table 3, as now described. In the column labeled B of Table 3,bold-faced type is used to indicate amino acid residues associated withenzyme recognition, normal-faced type is used to indicate protectiongroups for amino acids or peptides, and “•” is used to indicate apeptide scissile bond. Additionally, in the column labeled B, SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ IDNO: 6 are associated with certain of the amino acid residues shown inbold-faced type. As to each of SEQ ID NO: 5 and SEQ ID NO: 6, the first“Glu” or “E” of the sequence, may be a modifying group. The enzymaticcleavage site associated with the β-lactamase substrate moiety B is thelactam ring, the opening of which triggers the elimination of the DYE(comprising the -L-W moiety) from the 3′ position. In the columnslabeled “Cellular Localization Site of Enzyme Substrate,” “Cellular Siteof DYE or DYE-B′,” and “λ_(abs)/λ_(em) (nm/nm),” applicable conventionsare generally as previously described in relation to Table 2, althoughthe Cellular Site of DYE or DYE-B′ refer to the cellular site of theformer or the latter, and the λ_(abs)/λ_(em) refer to the wavelengthsfor the DNA-bound DYE or DNA-bound DYE-B′.

As previously described, substrates generally represented by Structure 1and Structure 2 are based on 3,8-diamino-6-phenylphenanthrodium dye.Other phenanthrodium dyes may be used to prepare enzyme substrates ofthe invention in any suitable manner, such as that described in relationto the 3,8-diamino-6-phenylphenanthrodium dye. The preparation and useof various phenanthrodium dyes, such as drug-related use, have beendescribed earlier. (Watkins, J. Chem. Soc. 3059 (1952); Watkins et al.,Nature 169, 506 (1952).)

A general representative structure for an enzyme substrate according toan embodiment of the invention is shown as Structure 3 below.

In the enzyme substrate generally represented by Structure 3 above, theDYE is an asymmetric cyanine-based nucleic acid dye. The dotted lineshown in Structure 3 represents the atoms associated with formation ofat least one fused aromatic ring, optionally comprising at least onenitrogen, which may or may not be quaternized. The represented atoms maybe those associated with the formation of at least one fused benzenering, merely by way of example. X may be O or S, for example. InStructure 3, n is 0, 1, 2 or 3, and d and ψ are as previously describedin connection with Structure 1 and Structure 2.

At least one ligand, but no more than two ligands, of ligands R₄, R₅, R₆and R₇ shown in Structure 3 may be represented as -L-W—B, where L, W andB are as described in relation to Structure 2. Merely by way of example,just one ligand of ligands R₄, R₅, R₆ and R₇, just one ligand of ligandsR₅ and R₇, or just ligand R₇ may be so represented as -L-W—B.

When R₄ is not -L-W—B, it may be H; a C1 to about C6 alkyl; a C1 toabout C6 alkoxy; a halogen; or an aryl meta to X, wherein the aryloptionally comprises at least one hetero atom of hetero atoms N, O andS. Merely by way of example, R₄ may be H; a methoxy meta to X; or anaryl meta to X, wherein the aryl optionally comprises at least onehetero atom of hetero atoms N, O and S. When R₅ is not -L-W—B, it may bea C1 to about C6 alkyl, such as a methyl, for example. When R₆ is not-L-W—B, it may be H; a C1 to about C10 alkyl, wherein the alkyloptionally comprises at least one hetero atom of hetero atoms N, O andS; a halogen; a C1 to C10 alkoxy or alkylmercapto, wherein the alkoxy orthe alkylmercapto optionally comprises at least one hetero atom ofhetero atoms N, O, and S; a C2 to about C12 dialkylamino, wherein thedalkylamino optionally comprises at least one hetero atom of heteroatoms N, O, and S; or a substituted or an unsubstituted aryl, whereinthe aryl optionally comprises 1 to 3 hetero atom(s) of hetero atomshalogen, N, O and S. When R₆ is not -L-W—B, it may be H; a C1 to aboutC6 alkyl; a C1 to about C6 alkoxy or alkylmercapto; a C2 to about C12dialkylamino, optionally comprising one N; or a substituted or anunsubstituted aryl, optionally comprising 1 to 3 hetero atom(s) ofhetero atoms N, O and S, merely by way of example. When R₇ is not-L-W—B, it may be H; a C1 to about C10 alkyl, optionally comprising anaryl and at least one hetero atom of hetero atoms N, O, and S; or asubstituted or an unsubstituted aryl, optionally containing 1 to 3hetero atom(s) of hetero atoms halogen, N, O, and S. When R₇ is not-L-W—B, it may be a C1 to about C3 alkyl, optionally comprising an aryl,merely by way of example.

Independently, each of R₈ and R₉ may be H; a C1 to about C10 alkyl,optionally comprising at least one hetero atom of hetero atoms N, O, andS; a halogen; a C1 to C10 alkoxy or alkylnercapto, wherein the alkoxy orthe alkylmercapto optionally comprises at least one hetero atom ofhetero atoms N, O, and S; a C2 to about C12 dialkylamino, optionallycomprising at least one hetero atom of hetero atoms N, O, and S; asubstituted or an unsubstituted aryl, optionally comprising 1 to 3hetero atom(s) of hetero atoms halogen, N, O and S. R₈ and R₉ may, incombination, form a fused aromatic ring, which may be furthersubstituted 1 to 4 time(s) independently by C1-C2 alkyl, C1-C2 alkoxy,C1-C2 alkylmercapto, or a halogen. Merely by way of example, R₈ and R₉are both H. Further, merely by way of example, R₅ and R₉, incombination, form a fused 6-membered ring, which may be furthersubstituted one time by a methyl, methoxy, methylmercapto, or a halogen.

According an embodiment of the invention, an enzyme substrate such asthat generally represented by Structure 3 above may comprise aglycosidyl, such as a glycosidyl enzyme substrate for a glycosidaseenzyme, for example.

A general representative structure for an asymmetric cyanine dye-basedenzyme substrate, according to an embodiment of the invention, is shownas Structure 4 below.

In the enzyme substrate generally represented by Structure 4 above, nmay be 0, 1, 2 or 3; n′ may be 1, 2, 3, 4 or 5; m′ may be 0 or 1; m″ is1, when m′ is O; m″ is 0, 2, 3 or 4, when m′ is 1; and ψ and d are asdescribed previously in relation to Structures 1, 2 and 3. Additionally,X may be O or S; each of R₈ and R₉ may be H, or R₈ and R₉, incombination, may form a fused benzene ring; and B and W are aspreviously described in relation to Structure 2.

Merely by way of example, a list of enzyme substrates that are generallyrepresented by Structure 4 above is provided in Table 4 of FIG. 12,along with information concerning same. Some general conventions applyto Table 4, as now described. The general conventions set forth inrelation to Table 3 generally apply to Table 4. In the column labeled Bof Table 4, when the substrate moiety molecule B is a peptide,bold-faced type is used to indicate amino acid residues associated withenzyme recognition and normal-faced type is used to indicate protectiongroups for amino acids or peptides. Additionally, in this column, SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 are associated with certain ofthe amino acid residues shown in bold-faced type. The enzymatic cleavagesite associated with the β-lactamase substrate moiety B is the lactamring, the opening of which triggers the elimination of the DYE(comprising the -L-W moiety) from the 3′ position. In the column labeled“Cellular Localization Site of Enzyme Substrate,” an indication of theprimary cellular location of the enzyme substrate, if the enzymesubstrate is applied to living cells, is provided. In this column,“membrane-impermeable” is indicated when the enzyme substrate isprimarily suitable for detecting enzyme activity in a cell-free system.In the column labeled “Cellular Site of DYE or DYE-B′,” an indication ofthe binding site of DYE or DYE-B′ following enzymatic cleavage isprovided. Generally, in a living-cell application, the primary cellularlocation of the DYE is the cell nucleus where the DNA resides. Finally,in the column labeled “λ_(abs)/λ_(em) (nm/nm),” the wavelengths providedrefer to the wavelengths for the DNA-bound DYE or DNA-bound DYE-B′.

A general representative structure for an asymmetric cyanine dye-basedenzyme substrate, according to an embodiment of the invention, is shownas Structure 5 below.

In the enzyme substrate generally represented by Structure 5 above, twosubstrate moiety molecules B are attached to an asymmetric cyanine dye.In Structure 5, n may be 0, 1, 2 or 3; n′ may be 1, 2, 3, 4, or 5; m′may be 0 or 1; when m′ is 0, m″ may be 1; when m′ is l, m″ may be 0, 2,3 or 4; q may be 1, 2, 3, 4 or 5; q′ may be 0 or 1; when q′ is 0, q″ is1; when q′ is 1, q″ may be 0, 2, 3 or 4; and ψ and d are as describedpreviously in relation to Structures 1, 2, 3 and 4. Additionally, X maybe O or S; each of R₈ and R₉ may be H, or R₈ and R₉, in combination, mayform a fused benzene ring; and B and W are as previously described inrelation to Structure 2. The enzyme substrate represented by Structure 5may comprise any of a number of suitable substrate moiety molecules B,such as any of those mentioned previously in relation to Structure 2,merely by way of example. Merely by way of example, the substrate moietymolecule B may comprise a peptide that comprises a fluorescencequencher, wherein the fluorescence quencher is attached to theN-terminal of an enzyme substrate moiety for an enzyme, such as acaspase enzyme, for example, and wherein the fluorescence quencher issufficient for removal from the dye upon enzymatic transformation of thesubstrate.

The enzyme substrate generally represented by Structure 5 may beparticularly suitable in terms of its two substrate moiety molecules B.While a single substrate moiety molecule B, when conjugated to anasymmetric cyanine dye, may be insufficient to reduce or to eliminatethe functionality of the dye, two such molecules may be sufficient inthis regard. When the enzyme substrate has two substrate moietymolecules B attached to the same nucleic dye, the nucleic acid-bindingaffinity of the dye may be significantly altered until both of themolecules are cleaved off the dye, which may result in a substantiallymore detectable (such as at least two times more detectable, forexample) fluorescent signal, or a positive signal of increasedfluorescence (such as at least two times more fluorescence, forexample). In general, if an enzyme substrate comprising a singlesubstrate moiety molecule B, such as that generally represented byStructure 4, results in a relatively poor signal-to-noise ratio, it maybe useful to employ an enzyme substrate having two substrate moietymolecules B, such as that generally represented by Structure 5, instead.

A general representative structure for an asymmetric cyanine dye-basedenzyme substrate, according to an embodiment of the invention, is shownas Structure 6 below.

In Structure 6, n, X, R₈, R₉, B, W, d and Ψ are generally as describedabove in relation to Structure 5. In the enzyme substrate generallyrepresented by Structure 6 above, two substrate moiety molecules B areattached to an asymmetric cyanine dye via a two-carbon and three-carbonlinker, respectively.

Merely by way of example, a list of enzyme substrates that are generallyrepresented by Structure 6 above is provided in Table 5 of FIG. 13,along with information concerning same. The general conventions thatapply to Table 5 are generally those previously described in relation toTable 3. All of the enzyme substrates provided in Table 5 may be usedfor enzyme activity detection in cell-free systems, such as celllysates, for example, and may be used for extracellular enzyme activitydetection, if present, in live cells, for example.

Enzyme Substrate Comprising Fluorescent Membrane Dye

A fluorogenic or fluorescent membrane dye may be a lipophilic dye thathas a high tendency to partition into a lipid environment, such as thatassociated with a liposome membrane or a cellular membrane. In general,a membrane dye is nonfluorescent or only weakly fluorescent in anaqueous phase and becomes highly fluorescent once in a membrane, suchthat specific membrane staining may result. Membrane dyes have been usedfor cell tracing, intracellular organelle mapping and cell membranerecycling studies. Generally speaking, membrane dyes may be roughlycategorized according to the mechanism or mechanisms by which theyinteract with a membrane. For example, a membrane dye may be roughly ashaving a membrane partition that is affected by membrane potential, oras having a membrane partition that is relatively unaffected by membranepotential. A dye in the former category is usually a small dye moleculethat carries a delocalized positive charge or a delocalized negativecharge, more typically a delocalized positive charge. Membrane stainingby such a dye largely relies on the electrostatic interaction betweenthe dye and the electrical field of the membrane. By way of example, amitochondrial dye carrying one delocalized cation may stain amitochondrial membrane in a membrane potential-dependent fashion. Theresponse of the staining to a membrane potential change may be a changein fluorescence intensity, a change in fluorescence wavelength, or acombination of both. A mitochondrial dye may be used to measuremitochondrial membrane potential, which may be an important indicator ofthe metabolic and/or health state of the mitochondria. A dye in thelatter category is usually a dye that is more hydrophobic than a dye inthe former category. Membrane staining by such a dye primarily involveshydrophobic interaction between the dye and the lipid environment of themembrane. Such a membrane potential-independent membrane dye may be anyof suitable such dyes, such as a cytoplasmic membrane dye, anendoplasmic reticulum (ER) dye, an endosome dye, a vacuole dye, asynaptic vesicle dye, and/or the like. Some of the more hydrophobicmitochondrial dyes are also known to stain mitochondria in a membranepotential-independent manner.

According to an embodiment of the invention, a substrate of theinvention DYE-(B)_(m) may comprise the DYE and at least one substratemoiety molecule B, wherein the subscript_(m) may be as previouslydescribed, such as 1, merely by way of example. In this embodiment, theDYE comprises a membrane dye, the at least one substrate moiety moleculeB comprises at least one highly water-soluble substrate moiety moleculeB, and the enzyme substrate DYE-(B)_(m) may be a weakly functionalmembrane dye. Merely by way of example, the enzyme substrate may haveincreased water-solubility or altered charge relative to the DYE untilenzymatic cleavage of the substrate, which may result in a highlyfunctional membrane product DYE or DYE-(B′)_(m), wherein B′ is a portionof B. The product DYE is formed when a scissile bond is between DYE andeach B. The product DYE-(B′)_(m) is formed when a scissile bond iswithin each B. Enzymatic cleavage of the substrate renders the membranedye functional, such that in the presence of a membrane, the functionaldye may partition into the membrane and a positive detectablefluorescence signal may be produced. In general, the positivefluorescence signal is increased at least about 2-fold relative to anybackground fluorescence associated with the starting enzyme substrate,such as at least about 10-fold or at least about 50-fold, merely by wayof example.

According to an embodiment of the invention, the substrate moietymolecule B may be any of a number of suitable substrate moieties, suchas a charged amino acid that is used with a peptidase, wherein the aminoacid is linked to the DYE via either C-linkage or N-linkage aspreviously described; a charged peptide that is used with a peptidase,wherein the peptide is linked to the DYE via either C-linkage orN-linkage as previously described and the peptide may optionallycomprise a fluorescence quencher that is removed from the DYE followingthe enzymatic transformation of the substrate; a substrate moiety thatis used with a β-lactamase; a phosphoryl that is used with an alkalineor an acid phosphatase; a sulfuryl that is used with a sulfatase; aβ-D-glucuronidyl that is used with a β-glucuronidase; aβ-D-galactopyranosidyl that is used with a β-D-galactosidase; anα-D-galactopyranosidyl that is used with an α-D-galactosidase; anα-D-mannopyranosidyl that is used with an α-D-mannosidase; anα-D-glucopyranosidyl that is used with an α-D-glucosidase; aβ-D-glucopyranosidyl that is used with a β-D-glucosidase; aX-β-D-cellobiosidyl that is used with a β-cellobiosidase; aN-acetyl-β-D-galactosaminidyl that is used with a neurominidase; aN-acetyl-β-D-glucosaminidyl that is used with aN-acetyl-β-D-glucosaminidase or a chitinase; a phosphorylated or anunphosphorylated phosphatidylinositol that is used with aphosphatidylinositol-specific phospholipase C; a glycosylatedphosphatidylinositol that is used with a phosphatidylinositol-specificphospholipase C; an adenosine-5′-phosphate that is used with aphosphodiesterase; a nucleoside-3′-phosphate that is used with anuclease or a ribonuclease; and/or the like, merely by way of example.

When the substrate moiety molecule B is a charged peptide, it maycomprise a peptide, a neutral segment of which may be involved in enzymeinteraction and another segment of which may be involved in modifying,such as increasing, for example, the water-solubility of the peptide.The modification may be facilitated by a modifying group of the lattersegment, whether original or added. Such modification may be useful tomodify a poorly soluble neutral peptide, such that it may be used in thepreparation of a membrane dye-based substrate of the invention. Forexample, a relatively water-insoluble tetrapeptide substrate moietyAla-Ala-Ala-Ala (SEQ ID NO: 4) for an elastase may be made morewater-soluble by attaching a highly water soluble modifier group. Such amodifier group may be any of suitable such groups, such as a poly-Asp, apoly-Glu, a poly-Lys, a poly-anionic nonpeptide group, a poly-cationicnonpeptide group, a water-soluble fluorescence quencher, and/or thelike, merely by way of example.

The DYE may comprise a mitochondrial dye and the scissile bond may besuch that following enzymatic cleavage the charge on the mitochondrialdye may be substantially unchanged and the mitochondrial dye may berendered functional. The scissile bond may be any of suitable scissilebonds, such as a bond derived from a substrate moiety that relies on ahydroxy functional group from the DYE, a peptide bond derived fromC-linked peptides and a low pKa amine (such as a pKa of less than 8, forexample) functional group from the DYE, and/or the like. According to anembodiment of the invention, the DYE may comprise a symmetrical cyanine,an asymmetrical cyanine, a merocyanine, a styryl dye, and/or the like.

A general representative structure for an enzyme substrate, according toan embodiment of the invention, is shown as Structure 7 below.

In the enzyme substrate generally represented by Structure 7, each of R₁and R₄, independently, may be H or Cl; R₂ may be a C1 to C12 alkyl, suchas a methyl or an ethyl, for example, a C7 to C12 arylalkyl, or a-L-W—B; each X and Y, independently, may be (CH₃)₂C, O, S, or NR₃, whereR₃ may be a C1 to C12 alkyl, a C7 to C12 arylalkyl, or a -L-W—B; n maybe 1, 2 or 3; and Ψ and d are as previously described. Additionally, Bcomprises a substrate moiety; L may be a C2 to C12 aliphatic linker,optionally comprising an aryl and at least one hetero atom of heteroatoms O and N; and W may be an atom or atoms O, C═O, NH or S, as may beassociated with the formation of a scissile bond between W and B for aspecific enzyme, or may be an atom or atoms O, C═O, NH or S, as may beassociated with the formation of a covalent linkage between B and theDYE, wherein the enzyme cleavage site is within B. Structure 7 comprisesno more that two -L-W—B.

According to an embodiment of the invention, in the enzyme substrategenerally represented by Structure 7, R₁ is C1; R₂ is a methyl; X isN—R₃, where R₃ is a benzyl; Y is (CH₃)₂C, O, or S; R₄ is H; each of n, dand Ψ are as described in relation to Structure 7; and -L-W—B isgenerally represented by Sub-structure 3 below.

In -L-W—B generally represented by Sub-structure 3, W and B are asdescribed in relation to Structure 7 above, and subscripts n′ and n″ aresuch that when n′ is 0, n″ may be 1 or 2, and when n′ is 1, n″ may be 0,1 or 2.

According to an embodiment of the invention, in the enzyme substrategenerally represented by Structure 7, R₁ is Cl; R₂ is a methyl; X isN—R₃, where R₃ is -L-W—B; Y is (CH₃)₂C, O, or S; R₄ is H; each of n, dand Ψ are as described in relation to Structure 7; and -L-W—B isgenerally represented by Sub-structure 3 above.

Merely by way of example, a list of enzyme substrates that are generallyrepresented by Structure 7 above is provided in Table 6 of FIG. 14,along with information concerning same. Some general conventions applyto Table 6, as now described. In the column labeled “Structure,”bold-faced type is used to indicate amino acid residues and SEQ ID NO: 1is associated with certain of the amino acid residues shown inbold-faced type. The general conventions that apply to the columnslabeled “Cellular Localization Site of Enzyme Substrate,” “Cellular Siteof DYE or DYE-B′,” and “λ_(abs)/λ_(em) (nm/nm),” Table 6 are generallythose previously described in relation to Table 3. All of the enzymesubstrates shown in Table 6 may be used in a cell-free system.

A membrane dye-based enzyme substrate of the invention may comprise astyryl dye and a substrate moiety molecule B. Such an enzyme substrate,according to an embodiment of the invention, may be generallyrepresented by Structure 8A shown below, according to an embodiment ofthe invention, or may be generally represented by Structure 8B shownbelow, according to another embodiment of the invention, merely by wayof example.

In the enzyme substrate generally represented by Structure 8A or byStructure 8B, f may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as 1, 2,3, 4, 5 or 6, for example; n may be 1, 2 or 3; n′ may be 1, 2, 3, 4 or5; m′ may be 0 or 1; when m′ is 0, m″ may be 1, 2, 3 or 4; when m′ is l,m″ may be 0, 2, 3 or 4; and each of W, B, ω and d is as previouslydescribed in relation to Structure 7.

Merely by way of example, a list of membrane dye-based enzyme substratesthat are generally represented by Structure 8A or Structure 8B above isprovided in Table 7 of FIG. 15, along with information concerning same.Some general conventions apply to Table 7, as now described. In thecolumn labeled “Structure,” bold-faced type is used to indicate aminoacid residues and SEQ ID NO: 1 is associated with a certain amino acidresidue shown in bold-faced type. The general conventions that apply tothe columns labeled “Cellular Localization Site of Enzyme Substrate,”“Cellular Site of DYE or DYE-B′,” and “λ_(abs)/λ_(em) (nm/nm)” of Table7 are generally those previously described in relation to Table 3. Allof the enzyme substrates shown in Table 7 may be used in a cell-freesystem.

Enzyme Substrate Comprising Other Functional Fluorescent Dye

Any of a number of enzyme substrates of the invention may be preparedfrom functional dyes, other than those described herein, in a manner orusing a method such as described herein. Any of a number of suitablefunctional dyes, such as a fluorescent dye-labeled receptor ligand, alysosome dye, a Golgi dye, and/or the like, merely by way of example.For example, a fluorescent lysosomal dye, such as any commerciallyavailable LysoTracker dye, may comprise a basic amine group. The basicamine group may be necessary to such a dye, as protonation of the aminegroup may serve to trap the dye in an organelle, such that specificlysosomal staining results. The amine group, such as a primary amine,for example, may be blocked with an amino acid substrate moiety or apeptide substrate moiety to form a peptidase substrate of the invention,which on cleavage may provide the lysosomal dye. Preparation of suchenzyme substrates may be understood in relation to the synthesis andexample descriptions provided below concerning similar substrates.

Enzyme Substrate Synthesis

The synthesis of an enzyme substrate of the invention is now generallydescribed. As previously described, such an enzyme substrate has a dyecomponent and a substrate moiety component, selection or preparation ofwhich is now described.

A functional fluorescent dye may be suitably functional to serve as acomponent of an enzyme substrate of the invention. For example, afunctional fluorescent dye may already possess a suitable functionalgroup, such as that sufficient for covalently connecting the dye to asubstrate moiety molecule B, for example. Merely by way of example, eachof a commercially available 9-amino-6-chloro-2-methoxyacridine and anethidium bromide has at least one aromatic amino group that may serve asa suitable functional group. A fluorescent dye that may not possesssuitable functionality may be derivatized for such functionality. Forexample, a fluorescent dye may derivatized such that is has a suitablefunctional group, such as that sufficient for covalently connecting thedye to a substrate moiety molecule B, for example. The functional groupmay be part of the chromophore core structure of the dye or may beattached to the dye via a linker molecule. The functional group may beany of a number of suitable functional groups, such as an amine group, acarboxylic group, a hydroxy group, and/or the like, merely by way ofexample. Any of various methods of derivatizing functional fluorescentdyes, such as any suitable method known in the art, may be employed.Merely by way of example, methods of preparing a fluorescent nucleicacid dye with a functional/reactive group have been disclosed in U.S.Pat. No. 5,863,753.

A substrate moiety B may be of suitable form to serve as a component ofan enzyme substrate of the invention. Merely by way of example, acommercially available glycosyl substrate moiety may be of suitableform, such as a suitably protected form, as provided. A substrate moietyB may not be of suitable form, as just described, such that it may haveto be prepared to have such form. For example, a substrate moiety B mayhave to be pre-assembled before it is coupled to the dye. Merely by wayof example, when the substrate moiety B is a peptide, the peptide may besynthesized before being coupled to the dye. Further, merely by way ofexample, the substrate moiety B may be rendered protected relative to asubsequent coupling reaction, before the coupling reaction. Suchprotection may be pursued, for example, when the coupling reaction maybe incompatible with one or more chemical group(s) of the substratemoiety B. Merely by way of example, in the case of a peptide substratemoiety B comprising amino acid residues with reactive side-chains, itmay be useful or necessary to protect the reactive side-chains, beforethe coupling reaction. Further, merely by way of example, in the case ofa glycosyl substrate moiety B having a hydroxyl and/or a carboxy, it maybe useful or advantageous to protect any hydroxy and/or any carboxy thatmay be present, before the coupling reaction. In general,suitably-protected glycosyl substrate moiety molecules having suitablereactive groups are commercially available from several sourcesincluding Sigma-Aldrich Chemicals Co. (St. Louis, Mo.), or can beprepared as generally known.

An enzyme substrate of the present invention may be prepared from asuitable functional fluorescent dye component comprising a suitablefunctional group and a suitable substrate moiety B component comprisinga suitable reactive group. These components may be obtained or prepared,as described above. These components are coupled via a couplingreaction. Any of various methods of coupling the components, such as anysuitable method known in the art, may be employed. Merely by way ofexample, a precursor peptide substrate moiety B may be coupled to afunctional dye to form a precursor peptidase substrate via any knownmethod of amide bond formation. Further, merely by way of example, aprecursor bromoglycosyl substrate moiety B may be coupled to afunctional dye having a hydroxy group to form a precursor glycosidasesubstrate by following a Koenigs-Knor procedure or another suitableprocedure. Still further, merely by way of example,phosphooxytrichloride may be coupled to a functional dye having ahydroxy group to form a precursor phosphatase substrate, which uponhydrolysis forms the final enzyme substrate.

A precursor substrate may be processed to provide a final enzymesubstrate. Such processing may involve hydrolysis, as in the case justdescribed, deprotection, and/or the like, merely by way of example. Forexample, a precursor peptidase substrate comprising one or moret-BOC-protected amine side-chain(s) or t-butyl ester-protectedcarboxylic acid side-chain(s) may be processed for deprotection usingtrifluoroacetic acid. Further by way of example, a precursor glycosidasesubstrate may be processed for deprotection, or the removal ofhydroxyl-protecting acetyl group(s), using sodium methoxide or sodiumethoxide.

Although a general synthesis procedure, such as any described above, maybe used to prepare an enzyme substrate of the invention, any of a numberof other suitable synthesis procedures may be employed. Merely by way ofexample, in a synthesis of a peptidase enzyme substrate, the dye may beconjugated to the C-terminal amino acid, and subsequently, amino acidresidues may be attached to the peptide substrate moiety B one at atime. Further, merely by way of example, a peptide substrate moiety B ora precursor peptide substrate moiety may be attached to a linkermolecule L, which itself comprises a reactive group, and subsequently,the resulting L-B conjugate may be covalently linked to the reactivegroup of a functional dye. In general, selection of an appropriate ordesirable preparation method may involve a consideration of the natureof the DYE, the nature of the substrate moiety B, and knowledge in theart.

Various asymmetric cyanine dyes comprising at least one reactive groupmay be prepared according to various reactions that are generallyrepresented in Panel 1 of FIG. 16. Additional information concerning thepreparation of various asymmetric cyanine dyes may be found in U.S. Pat.Nos. 5,321,130, 5,436,134 and 5,863,753, and in Brooker et al., J. Am.Chem. Soc. 64, 199 (1942).

Various phenanthrodium dyes comprising at least one reactive group maybe prepared according to various reactions that are generallyrepresented in Panel 2 of FIG. 17. Additional information concerning thepreparation of various phenanthrodium dyes may be found in U.S. Pat. No.5,437,980 and references identified therein.

Various cyanine-based mitochondrial dyes comprising at least onereactive group may be prepared according to various reactions that aregenerally represented in Panel 3 of FIG. 18. Additional informationconcerning the preparation of various cyanine-based dyes may be found inreferences such as those mentioned in relation to Panel 1.

Various styryl-based mitochondrial dyes comprising at least one reactivegroup may be prepared according to various reactions that are generallyrepresented in Panel 4 of FIG. 19. Additional information concerning thepreparation of various styryl-based dyes may be found in Hassner et al.,J. Org. Chem. 49, 2546 (1984).

Various peptidase substrates may be prepared using nucleic dye ormitochondrial dye according to various reactions that are generallyrepresented in Panel 5 of FIG. 20. Panel 5 includes a legend thatreferences Asp-Glu-Val-Asp or DEVD (SEQ ID NO: 1) andAsp(OBu-t)-Glu(OBu-t)-Val-Asp(OBu-t) or D(OBu-t)-E(OBu-t)-V-D(OBu-t)(SEQ ID NO: 12). The peptide substrate moietyAc-Asp(OBu-t)-Glu(OBu-t)-Val-Asp(OBu-t)-OH, orAc-D(OBu-t)-E(OBu-t)-V-D(OBu-t)-OH, where D(OBu-t)-E(OBu-t)-V-D(OBu-t)is associated with SEQ ID NO: 12, may be synthesized using techniquesemployed in peptide chemistry, or may be purchased from a commercialsource (Zhang et al., Bioconjugate Chem. 14, 458 (2003). Additionalinformation concerning the preparation of various styryl-based dyes maybe found in Hassner et al., J. Org. Chem. 49, 2546 (1984).

Various peptidase substrates may be prepared using a reaction that isgenerally represented in Panel 6 of FIG. 21. Panel 6 includes a legendthat references Leu-Glu-Glu-Asp or LEED (SEQ ID NO: 3) andLeu-Glu(OBu-t)-Glu(OBu-t)-Asp(OBu-t) or L-E(OBu-t)-E(OBu-t)-D(OBu-t)(SEQ ID NO: 13).

According to the reaction generally represented in Panel 6, a precursorpeptide substrate moiety B may be conjugated to a mono-protectedbifunctional linker molecule and the resulting linker-precursor B may bedeprotected and coupled to a dye, as generally illustrated. This method,using a common linker-precursor B, allows one to quickly preparemultiple enzyme substrates from dyes of different colors andfunctionalities.

A glycosidase substrate of the invention may be prepared from afunctional dye comprising at least one hydroxy group and a suitablehalocarbohydrate molecule, such as a bromocarbohydrate molecule, forexample, in the presence a catalyst. Methods for conjugating ahalocarbohydrate to a hydroxy compound to form a glycosidic linkage havebeen described in U.S. Pat. Nos. 5,030,721 and 5,208,148, and in otherpublications (Rotman et al., Proc. Natl. Acad. Sci. USA 50, 1 (1963);Fernández-Santana et al., Glycoconjugate J. 15, 549 (1998); K{hacek over(r)}en et al., J. Chem. Soc. Perkin Trans. 1, 2467 (1997)). By way ofillustration, the synthesis of a β-D-glucuronidase substrate from amitochondrial dye may be carried out via a reaction that is generallyrepresented in Panel 7 of FIG. 22.

Preparations of other enzyme substrates of the invention will beunderstood from the description herein and/or knowledge in the art.Merely by way of example, a phosphatase substrate of the invention maybe prepared from a functional dye with a hydroxy group andphosphooxytrichloride by following a procedure described by Rotman etal., Proc. Natl. Acad. Sci. USA 50, 1 (1963); a sulfatase substrate ofthe invention may be prepared from a functional dye with a hydroxy groupand chlorosulfonic acid by following a procedure of Scheigetz et al.,Organic Prep. Proc. Int. 29, 561 (1997); and aphosphatidylinositol-specific phospholipase C substrate may be preparedas described in any of several publications, namely, Birrell et al.,Biophys. J. 84, 3264 (2003); Zaikova et al., Bioconjugate Chem. 12, 307(2001); and Rukavishnikov et al., Bioorg. Med. Chem. Lett. 9, 1133(1999).

Enzyme Substrate Applications

An enzyme substrate of the invention that is specific for an enzyme orenzymes may be used to detect the activity of the enzyme or enzymes in acell-free biological sample. In such detection, a substrate of theinvention specific for the enzyme(s) and a suitable partner molecule,partner molecules, or a suitable assembly of partner molecules, whichmay be bound by the released functional dye, DYE or DYE-(B′)_(m), may beselected. The enzyme substrate may be placed, or dissolved, in asuitable buffer or a water-miscible organic solvent. The partnermolecule, partner molecules, or assembly of partner molecules may beplaced, or dissolved, in a suitable buffer. The resulting solutions maythen be combined with a biological sample containing the enzyme.Fluorescence of the resulting combination may then be detected using adetection device, such as a fluorometer or a fluorescence microplatereader, for example, at wavelengths specific to the DYE or DYE-(B′)_(m).Alternatively, the enzyme substrate and the binding molecule or assemblyof binding molecules may be combined in a suitable biological buffer,and the resulting combination may then be combined with the biologicalsample, whereupon fluorescence may be detected. In either case, thebinding molecule or the assembly of binding molecules may be in anamount sufficient to saturate the functional dye, DYE or DYE-(B′)_(m),when the enzyme substrate is completely hydrolyzed.

The substrate moiety B alone or a combination of the substrate moiety Band the linkage between it and the DYE may provide the enzyme substratewith specificity for the particular enzyme(s), while the partnermolecule, partner molecules, or assembly of partner molecules mayprovide the enzymatically-released DYE or DYE-(B′)_(m) with the bindingsite or sites. When the detection concerns enzyme activity in acell-free sample, the released functional dye, DYE or DYE-(B′)_(m), maygenerally be a nucleic acid dye or a membrane dye, such as amitochondrial dye or a cytoplasmic membrane dyes, for example. Thebinding molecule or assemblies of binding molecules may be nucleicacid(s), liposome(s), or micelle(s). According to an embodiment of theinvention, the DYE or DYE-(B′)_(m) may be a nucleic acid dye and thebinding molecule(s) may be nucleic acid(s), such as DNA molecule(s).Merely by way of example, a cell-free biological sample may be a celllysate or a solution comprising, or believed to comprise, an enzyme tobe detected without other cellular components from lysed cells.

Certain enzymes may be used in immunohistochemical techniques orenzyme-linked immunosorbent assays (ELISAs). In applications such asthese, a fluorogenic, a chromogenic, or a chemiluminogenic enzymesubstrate may be used in conjunction with an enzyme-conjugated secondarydetection reagent for highly sensitive detection of an analyte.According to an embodiment of the invention, an enzyme substrate of theinvention in combination with an excess of a partner molecule, partnermolecules, or an assembly of partner molecules may be used in an ELISAapplication. Merely by way of example, the enzyme substrate may be anucleic acid dye-based glycosidase substrate, or a nucleic aciddye-based alkaline phosphatase substrate.

An enzyme substrate of the invention that is specific for an enzyme orenzymes may be used to detect the intracellular or extracellularactivity of the enzyme or enzymes in a living cell culture or livingcell tissue. In such detection, a stock solution of the enzyme substratein a buffer or a water-miscible organic solvent may be added to astandard cell culture or tissue culture. The buffer may be any of anumber of suitable buffers, such as a PBS buffer, a Tris buffer, and/orthe like. The water-miscible organic solvent may be any of a number ofsuitable such solvents, such as DMSO, DMF, methanol, ethanol, and/or thelike. When an organic solvent is used, the stock solution should be of aconcentration sufficient such that upon its dilution in the culturemedium, the organic solvent does not interrupt cell membranes. Merely byway of example, the amount of organic solvent in the final culturemedium may be about 2% or less, or about 1% or less. The cells may beincubated, such that the substrate may enter the cells and react withthe enzyme. The reaction may be for a sufficient amount of time, such ason the order of about 5 minutes to about 10 hours, for example.Fluorescence may be detected via analysis of the cell culture or thetissue culture via a fluorescence microscope, a fluorescence microplatereader, a flow cytometer, and/or the like, for example. The fluorescencesignal may be in the form of a change in fluorescence signal intensityat a wavelength specific to the functional dye, DYE, a fluorescencewavelength shift, a physical re-distribution of fluorescence signalwithin the cells, and/or the like, or any combination thereof, forexample. In general, the enzymatically-released DYE or DYE-(B′)_(m) mayform a cellular staining pattern that is characteristic of the cellulardistribution of the partner molecule, partner molecules, and/or assemblyof partner molecules. In general, washing the cell sample with a normalculture medium before examination may reduce background fluorescencefrom broken cells or decomposed enzyme substrate.

According to an embodiment of the invention, intracellular enzymeactivity may be detected using an enzyme substrate of the invention thatcomprises a functional dye that is a nucleic acid dye, a mitochondrialdye, a cytoplasmic membrane dye, and/or the like. According to anembodiment of the invention, extracellular enzyme activity may detectedusing an enzyme substrate of the invention that comprises a functionaldye, such as a membrane dye, and/or the like. The extracellular enzymemay be any suitable such enzyme, such as a gelatinase, a collagenase, amatrix metalloproteinase (MMP), and/or the like, for example.

Biological characteristics of cells and living species may be associatedwith the selective regulation of gene expression as a result ofintrinsic developmental programs and extrinsic signals. Studies of geneexpression regulation may be conducted using so-called reporter genes,which encode for easily detectable protein products, such as afluorescent protein or an enzyme, or so-called reporter enzyme. Areporter enzyme may be detected bioluminescently or fluorescentlydepending on the nature of the enzyme. Firefly luciferase and Renilaluciferase, for example, are each detected using an enzyme-specificsubstrate, D-luciferin and coelenterazine, respectively, which isenzymatically oxidized to produce light. Other reporter enzymes, such asβ-lactamase, β-galactosidase and β-glucuronidase, may be detectedfluorescently using a fluorogenic enzyme substrate specific for theenzyme. Numerous fluorogenic enzyme substrate substrates have beendeveloped for reporter enzymes. These existing fluorogenic enzymessubstrates of prior art, when applicable, release a fluorescent productthat is passively trapped within cells, confined by cytoplasmicmembranes. In general, the fluorescent products from these enzymesubstrates merely indicate the presence of certain enzyme activity, butno additional cellular information.

According to an embodiment of the invention, an enzyme substratecomprises a functional dye that, following enzymatic cleavage, binds toa partner molecule, partner molecules, or an assembly of partnermolecules. An enzyme substrate of the invention may be used in areporter gene assay, wherein the reporter gene encodes for a reporterenzyme that hydrolyzes the substrate to release a functional dye, DYE orDYE-(B′)_(m). In general, the resulting functional dye, DYE orDYE-(B′)_(m), is not fluorescent until it binds to a partner molecule,such as DNA or RNA, for example, partner molecules, or an assembly ofbiological partner molecules, such as cell membranes or cellularorganelles, for example. The functional dye, DYE or DYE-(B′)_(m), mayprovide additional cellular information by indicating the physicaldistribution, quantities, morphology, and/or the like, of the cellularcomponents. According to an embodiment of the invention, an enzymesubstrate of the invention that may be used for gene expression assaysmay be a functional dye, such as a mitochondrial dye, a lysosomal dye,an endosomal dye, a cytoplasmic membrane dye, a nucleic acid dye, and/orthe like, by way of example. According to an embodiment of theinvention, the functional dye may be a mitochondrial dye, a cytoplasmicmembrane dye, or a nucleic acid dye. The enzyme substrates of theinvention that may be used for gene expression assays may be a substratefor β-lactamase encoded by a β-lactamase gene, a β-galactosidase encodedby a lacZ gene, a β-glucuronidase encoded by a GUS gene, and/or thelike, for example. According to an embodiment of the invention, acombination of an enzyme substrate of the invention and a reporter genethat encodes for a reporter enzyme may be used for high throughputscreening of drug candidates, such as those whose therapeutic mechanismmay involve intervening in the expression of a certain gene.

According to an embodiment of the invention, a method for determiningthe effect of a test substance that may be exogenously added to anenzyme is provided. In this method, the enzyme may be involved in abiological process in a test cell, the test substance may be exogenouslyadded to the enzyme, and the effect of the test substance may bedetermined. Generally, such a method may comprise contacting the testcell with the test substance and the enzyme substrate of the inventionthat is specific for the enzyme. This may occur under variousconditions, such as a condition in which the test substance interactswith an external membrane receptor of the cell or a condition in whichthe test substance and the enzyme substrate enter the cell, for example.The fluorescence of the text cell may be detected or recorded. Thefluorescence of a control cell, which has been in contact with theenzyme substrate, but not the test substance, may also be detected orrecorded. If a comparison of the fluorescence associated with the testcell and the control cell shows a difference in fluorescence, such as adifference in fluorescence intensity, fluorescence wavelength, orphysical distribution, for example, such a difference may be indicativeof an effect of the test substance on the enzyme. A similar method maybe carried out using a reference substance that is known to have aneffect on the enzyme. Comparison of the results obtained using the testsubstance and the reference substance may be used to evaluate,quantitatively or qualitatively, the relative effectiveness of the testsubstance.

According to an embodiment of the invention, an enzyme substrate of theinvention that comprises a near infrared (near IR) dye, DYE, may be usedfor real-time in vivo imaging of any of a variety of clinically relevanttargets. Generally, near IR dyes are dyes having wavelengths from about600 nm to about 1300 nm. Merely by way of example, a suitable near IRdye, DYE, may have an absorption wavelength and an emission wavelengthfrom about 630 to about 1200 nm. Near IR light penetrates tissuesrelatively easily (Wyatt et al., Phil. Trans. R. Soc. London B 352, 701(1997)), such that a fluorescent IR dye may be used for optical imagingof internal tissue in a living animal, such as a human, for example.Near IR fluorescent imaging may be advantageous relative to otherclinical imaging techniques for any of a number of reasons, such asmulticolor imaging capability, high temporal and special resolution,avoidance of hazardous ionizing radiation, improved safety, and/or thelike, for example. According to various embodiments of the invention, anenzyme substrate of the invention comprising an IR dye, DYE, may be usedfor, respectively, in vivo detection of enzyme activity in disease, invivo monitoring of the efficacy of an inhibitor or an activator of anenzyme involved in a biological process, in vivo imaging of geneexpression, and guiding a surgical intervention.

According to an embodiment of the invention, at least two enzymesubstrates of the invention may be applied to a living cell to detectthe same intracellular enzyme. In this embodiment, the substrate moietymolecules B of the enzyme substrates are specific for the same enzyme,while the functional dyes of the enzyme substrates are functionallydifferent, such that when released, the functional dyes detect differentcomponents of the cell. Using such a method of intracellular enzymedetection, one may detect a particular intracellular enzyme andvisualize multiple cellular components under a fluorescent microscope.For example, when the functionally different dyes have differentfluorescence colors, multiple cellular components may be identifiedunder a fluorescence microscope by their colors and their morphologies.Further by way of example, when the functionally different dyes havesimilar absorption and emission spectra, a single excitation may exciteall of the functionally different dyes and the fluorescence emissionsfrom all of these functionally different dyes may be collected withinthe same optical window. In general, a method of intracellular enzymedetection that employs multiple enzyme substrates, such as any of thosejust described, will produce a greater amount of fluorescence signalrelative to a method that employs one enzyme substrate of the invention.This may be explained by the fact that in the former case, the maximalamount of fluorescence signal is not limited by the abundance of asingle cellular component, as it is in the latter case.

According to an embodiment of the invention, at least two enzymesubstrates of the invention may be applied to a living cell to detectthe same number of intracellular enzymes. In this embodiment, eachsubstrate moiety B of an enzyme substrate is specific for a particularenzyme, while the functional dyes of the enzyme substrates arefunctionally different, such that when released, the functional dyesdetect different components of the cell. The functional dyes may bespectrally distinct from one another. In such a case, the presence orabsence of each particular enzyme may be indicated by both thefluorescence intensity at a wavelength unique to each functional dye andby the staining of the cellular component by each functional dye.

Examples concerning enzyme substrates of the invention, and associatedmethods of preparation or use of same are provided below. These examplesare illustrative, not limiting, as to any aspect, feature, embodiment,and/or the like, of the present invention. Other suitable modificationsand adaptations of various conditions and parameters, such as thosenormally encountered in in vitro or in vivo assays, drug screeningprocedures, diagnostic procedures, and/or the like, will be appreciatedas being within the spirit and scope of the invention.

EXAMPLES

Unless otherwise noted, all chemical materials used for syntheses wereof at least reagent grade and were purchased from either AldrichChemicals Co. (Milwaukee, Wis.) or VWR International (West Chester,Pa.).

Example 1 Preparation of Compound No. 1

2-Methylbenzothiazole (4.51 g, 30 mmoles) and methyl p-toluenesulfonate(5.62 g, 30 mmoles) were mixed in a 50 mL round-bottom flask and heatedat 110° C. for 24 hours. The resulting solid was triturated with ether(100 mL) and then collected by suction filtration. The collected solidwas dried under vacuum at room temperature for 24 hours to give CompoundNo. 1, namely, 2,3-dimethylbenzothiazolium p-toluenesulfonate.

Example 2 Preparation of Compound No. 2

A suspension of Compound No. 1 (1.1 g, 3.28 mmoles) andN,N′-diphenylformamidine (1 g, 5.1 mmoles) in Ac₂O (3 mL) was stirred atabout 110° C. for about 1 hour. The reaction mixture was cooled to roomtemperature and then poured into ether (50 mL). The solid was collectedfrom the solution by suction filtration and then dried under vacuum togive Compound No. 2, as generally represented by the structure below.

Example 3 Preparation of Compound No. 3

A mixture of lepidine (5 g) and 4-bromobutyric acid ethyl ester (5equivalents) was heated at 130° C. for 24 hours. The oily product wasthoroughly triturated with ethyl acetate, suction filtered and thendried under vacuum to give the Compound No. 3, as generally representedby the structure below.

Example 4 Preparation of Compound No. 4

Compound No. 4 was prepared from lepidine (5 g) and of 6-bromohexanoicacid (5 equivalents), using the general procedure described above inrelation to Example 3.

Example 5 Preparation of Compound No. 5

A mixture of 2-methylmercaptobenzothiazole and an equal molar amount ofmethyl p-toluenesulfonate was heated at 120° C. overnight. The resultingsolid was crushed and briefly stirred in ethyl acetate. The resultingsolid was collected from the resulting mixture by filtration and thendried under vacuum for 24 hours to give Compound No. 5, namely,3-methyl-2-methylmercapto-benzothiazolium p-toluenesulfonate.

Example 6 Preparation of Compound No. 6

A mixture of Compound No. 4 (1 g) and an equal molar amount of CompoundNo. 5 were dissolved in dry DMF (15 mL) to give a solution. Threeequivalents of triethylamine were added to the solution, and theresulting solution was stirred at room temperature overnight. Theresulting orange solution was poured into a solution of NaI (10equivalents) dissolved in water (100 mL). The resulting mixture wasbriefly stirred and suction filtered to collect the product, which wasthen dried under vacuum at 45° C. for 24 hours. The crude product waspurified on a silica gel column eluted with MeOH/CHCl₃ (5%-20%) to giveCompound No. 6, as generally represented by the structure below, whichwas an orange-colored solid. Compound No. 6 is an example of a nucleicacid dye comprising a carboxylic acid functional group.

Example 7 Preparation of Compound No. 7

A solution of Compound No. 6 (1 g) dissolved in dry DMF (25 mL) wasprepared. An equivalent of each of triethylamine and TSTU were addedsuccessively to the solution. After about 20 minutes of stirring, anequivalent of mono-t-Boc-ethylenediamine (Quanta Biodesign, Ltd.,Powell, Ohio) in DMF (2 mL) was added dropwise. The solution wascontinuously stirred at room temperature for about 3 to about 4 hoursand then poured into a solution of NaI (10 equivalents) in water (150mL). A solid was collected from the resulting solution by suctionfiltration and then dried under high vacuum for 24 hours at 45° C. Thesolid was resuspended in dichloromethane (20 mL) and stirred in anice/water bath. Trifluoroacetic acid (about 5 mL) was added dropwise tothe stirred solution. Stirring continued until TLC (20%MeOH/CHCl₃/silica plate) showed completion of the deprotection reaction(about 2 to about 3 hours). The solvent was removed by rotaryevaporation. A resulting gummy solid was triturated repeatedly withether until an orange chunky solid was obtained. This solid was isolatedby centrifugation and then dried under high vacuum at room temperatureto give Compound No. 7, as generally represented by the structure below.Compound No. 7 is an example of a nucleic acid dye comprising an aminefunctional group.

Example 8 Preparation of Compound No. 8

A solution of Fmoc-Asp(OBu-t)OH (366 mg, 0.89 mmole) in dry DMF (about 6mL) cooled in an ice/water bath was prepared and magnetically stirred.Triethylamine (124 μL, 0.89 mmole) and HBTU (340 mg) (Dublin, Calif.)were added successively to the stirred solution. After one hour ofstirring at 0-4° C., the solution was added portion-wise to a stirredsolution of Compound No. 7 (400 mg) in pyridine. The resulting solutionwas stirred until TLC (10% MeOH/CHCl₃/silica plate) showed completion ofthe coupling reaction (about 1 hour). The solution was added to astirred solution of NaI (4 g) in water (120 mL). A precipitate wascollected from the solution and dried under vacuum for 24 hours. Thecrude product was purified on a silica gel column eluted with MeOH/CHCl₃(5%-15%). The purified Fmoc-protected compound was suspended in CHCl₃ (8mL). Piperidine (about 2 mL) was added to the suspension and theresulting solution was stirred until the deprotection was complete asshown by TLC (about 2 hours). The solvent and excess piperidine wereremoved by rotary evaporation to give a pure amino intermediate. Thisintermediate was coupled to the three remaining Fmoc-protected aminoacids (Fmoc-Val-OH, Fmoc-Glu(OBu-t)OH and Fmoc-Asp(OBu-t)OH), one at atime, using the same procedure used for coupling the first amino acidFmoc-Asp(OBu-t)OH, to give Compound No. 8, as generally represented bythe structure below.

Example 9 Preparation of Compound No. 9

A solution of Compound No. 8 dissolved in DMF (5 mL) and pyridine (3 mL)was prepared and stirred. Acetic anhydride (about 1.5 mL) was addeddropwise to the stirred solution. After overnight stirring, the solutionwas poured into a solution of NaI (3 g) in water (100 mL). A precipitatewas collected from the mixture by suction filtration and then driedunder high vacuum for 24 hours at room temperature. The crude productwas purified on a silica gel column eluting with MeOH/CHCl₃ (5-15%) togive Compound No. 9, as generally represented by the structure below.

Example 10 Preparation of Substrate No. 19

A suspension of Compound No. 9 (0.35 g) in CH₂Cl₂ (16 mL) was preparedand stirred in an ice/water bath. Trifluoroacetic acid (50%, 8 mL) wasadded to this suspension. The resulting solution was continuouslystirred for one hour at 0-4° C. and then 5 hours at room temperature.The solvent was removed from the solution by rotary evaporation and theresidue was triturated with ether three times. The resulting solid wasisolated by centrifugation and then dried under vacuum to give CompoundNo. 19, as generally represented by the structure below. HPLC of thisproduct indicated 95% purity. Substrate No. 19 and informationconcerning same may be found in Table 4.

Example 11 Preparation of Compound No. 10

A solution of Fmoc-Asp(OBu-t)OH (27.3 g, 66.4 mmoles) in dry DMF (200mL) was prepared and stirred at 0-4° C. Small portions of triethylamine(9.2 mL, 66.4 mmoles) were added to the solution, followed by aportion-wise addition of TSTU (20 g, 66.4 mmoles). After one hour ofcontinued stirring, the solution was added portion-wise to a stirredsuspension of Glu(OBu-t)OH (13.5 g, 66.4 mmoles) (Advanced ChemTech,Inc., Louisville, Ky.) in DMF (75 mL) and pyridine (75 mL). Theresulting mixture slowly turned into a homogenous solution on continuedstirring. After about 24 hours of stirring at room temperature, thesolvent was removed from the solution under high vacuum at roomtemperature. A solution of citric acid monohydrate (about 14 g) in water(about 300 mL) was added to the resulting residue. The resulting mixturewas thoroughly mixed and then extracted with ethyl acetate (about 500mL). The ethyl acetate layer was washed with water (2×150 mL) and brine(150 mL) and then dried with anhydrous sodium sulfate. Evaporation ofthe solvent gave a white solid dipeptide compound, Compound No. 10,namely, Fmoc-Asp(OBu-t)-Glu(OBu-t)-OH.

Example 12 Preparation of Compound No. 11

Fmoc-Asp(OBu-t)OH (10.17 g) was activated using triethylamine and HBTUas generally described above in relation to Example 8 and then reactedwith CBZ—NHCH₂CH₂NH₃ ⁺Cl⁻ in the presence of triethylamine (1equivalent) in DMF using standard peptide coupling conditions. TLCshowed that the reaction was complete in about 4 hours. The resultingmixture was poured into water (500 mL). A resulting solid was collectedfrom the resulting solution by suction filtration and then redissolvedin ethyl acetate (about 300 mL). The ethyl acetate solution was washedwith brine (200 mL) and then dried with anhydrous Na₂SO₄. Followingrotary evaporation of ethyl acetate, the Fmoc-protected compound wasredissolved in CHCl₃ (about 100 mL) and then deprotected with piperidineusing the general procedure described above in relation to Example 8.Evaporation of the solvent and excess piperidine gave Compound No. 11,namely, H-Asp(OBu-t)-NHCH₂CH₂NH—CBZ.

Example 13 Preparation of Compound No. 12

Compound No. 11 and Fmoc-Val-OH were coupled and the resulting conjugatewas deprotected to give a crude product using the general proceduredescribed above in relation to Example 8. The product was furtherpurified on a silica gel column eluted with MeOH/EtOAc (5%) to giveCompound No. 12, namely, H-Val-Asp(OBu-t)-NHCH₂CH₂NH—CBZ.

Example 14 Preparation of Compound No. 13

Compound No. 10 and Compound No. 12 were coupled and the resultingconjugate was deprotected to give a crude product using the generalprocedure described above in relation to Example 8. The product wasfurther purified on a silica gel column eluted with MeOH/CHCl₃ to giveCompound No. 13, namely,H-Asp(OBu-t)-Glu(OBu-t)-Val-Asp(OBu-t)-NHCH₂CH₂NH—CBZ, orH-D(OBu-t)-E(OBu-t)-V-D(OBu-t)-NHCH₂CH₂NH—CBZ, whereD(OBu-t)-E(OBu-t)-V-D(OBu-t) is associated with SEQ ID NO: 12.

Example 15 Preparation of Compound No. 14

Compound No. 14, namely,Ac-Asp(OBu-t)-Glu(OBu-t)-Val-Asp(OBu-t)-NHCH₂CH₂NH—CBZ, orAc-D(OBu-t)-E(OBu-t)-V-D(OBu-t)-NHCH₂CH₂NH—CBZ, whereD(OBu-t)-E(OBu-t)-V-D(OBu-t) is associated with SEQ ID NO: 12, wasprepared from Compound No. 13 using the general procedure describedabove in relation to Example 9.

Example 16 Preparation of Compound No. 15

Compound No. 14 was hydrogenated in MeOH using 5% Pd/C (5%) to giveCompound No. 15, namely,Ac-Asp(OBu-t)-Glu(OBu-t)-Val-Asp(OBu-t)-NHCH₂CH₂NH₂, orAc-D(OBu-t)-E(OBu-t)-V-D(OBu-t)-NHCH₂CH₂NH₂, whereD(OBu-t)-E(OBu-t)-V-D(OBu-t) is associated with SEQ ID NO: 12.

Example 17 Preparation of Compound No. 16

Compound No. 2 and Compound No. 3 were coupled using the generalprocedure described above in relation to Example 6 to give an ethylester dye intermediate, which without further purification washydrolyzed to a free acid using NaOH/H₂O/MeOH at room temperature. Theproduct was isolated by precipitation using HCl and then purified on asilica gel column eluted with MeOH/CHCl₃ (5-15%) to give Compound No.16, as generally represented by the structure below. Compound No. 16 isan example of a functional dye comprising a carboxylic acid functionalgroup.

Example 18 Preparation of Compound No. 17

A solution of Compound No. 16 (95 mg, 0.18 mmol) in DMF (3 mL) at roomtemperature was prepared. Et₃N (30 μL, 0.22 mmol) and TSTU (54 mg, 0.18mmol) were added to the solution. The resulting mixture was stirred atroom temperature for 30 minutes and Et₃N (50 μL) and Compound No. 15(11.7 mg, 0.16 mmol) were then added. The resulting mixture was stirredat room temperature overnight and then concentrated to dryness in vacuo.Trituration of the residue with CH₃CN gave a dark blue solid (170 mg),Compound No. 17, as generally represented by the structure below.

Example 19 Preparation of Substrate No. 20

A suspension of Compound No. 17 (100 mg, 0.09 mmol) in CH₂Cl₂ (8 mL) at5° C. was prepared. TFA (2 mL) was added to the suspension. Theresulting mixture was stirred at 5° C. for one hour and then at roomtemperature overnight. The resulting solution was concentrated todryness in vacuo. The resulting residue was triturated with CH₃CN togive a dark blue solid enzyme substrate (55 mg), Substrate No. 20, asgenerally represented by the structure below. Substrate No. 20 andinformation concerning same may be found in Table 4.

Example 20 Preparation of Compound No. 18

A mixture of Compound No. 1 (1.1 g, 3.28 mmoles) and malonaldehydebis(phenylimine) monohydrochlride (0.85 g, 3.28 mmoles) in aceticanhydride (15 mL) was refluxed for 1 hour and then cooled to roomtemperature. The mixture was evaporated to dryness under high vacuum.The resulting residue was purified on a short silica gel column elutedwith MeOH/CHCl₃ (2-10%) to give Compound No. 18, as generallyrepresented by the structure below.

Example 21 Preparation of Compound No. 19

Compound No. 19, as generally represented by the structure below, wasprepared from lepidine and bromoacetic acid using the general proceduredescribed above in relation to Example 3.

Example 22 Preparation of Compound No. 20

Compound No. 20, as generally represented by the structure below, wasprepared from Compound No. 18 and Compound No. 19 using the generalprocedure described above in relation to Example 6. Compound No. 20 isan example a near IR asymmetrical cyanine dye having a carboxylic acidfunctional group.

Example 23 Preparation of Compound No. 21

A solution of BOC-Lys(Ac)—OH (100 mg, 346.8 mmol) in DMF at 5° C. wasprepared. Et₃N (51 μL, 364.1 mmol) and TSTU (109 mg, 364.1 mmol) wereadded to the solution. The resulting mixture was stirred at 5° C. for 30minutes and Et₃N (100 μL) and Compound No. 6 (100 mg, 148.3 mmol) werethen added. The resulting mixture was stirred at 5° C. for 2 hours andthen at room temperature for one hour. The solution was poured into asolution of NaI (0.5 g) in water (50 mL). A solid was collected from themixture and then purified by column chromatography to give a dark bluesolid (37.9 mg), Compound No. 21, as generally represented by thestructure below. Compound No. 21 is an example of a substrate for HDAC.

Example 24 Preparation of Compound No. 22

Compound No. 22 (98.1 mg), as generally represented by the structurebelow, was prepared from BOC-Lys(FMOC)—OH (128 mg, 426.8 mmol) andCompound No. 6 (125.8 mg, 186.6 mmol) using the general proceduredescribed above in relation to Example 23.

Example 25 Preparation of Compound No. 23

A solution of Compound No. 22 (90 mg) in CHCl₃ (2 mL) was prepared.Piperidine (100 μL) was added to the solution. The resulting mixture wasstirred at room temperature for 2 hours and then concentrated to drynessin vacuo. The resulting residue was stirred as a suspension in EtOAc (5mL) for one hour and a resulting precipitate (20 mg) was collected bysuction filtration to give Compound No. 23, as generally represented bythe structure below.

Example 26 Preparation of Substrate No. 5 and Substrate No. 6

A solution of Z-Ala-Ala-OH (600 mg, 2 mmol) in DMF (5 mL) and pyridine(5 mL) at 0° C. was prepared. EDAC (410 mg, 2.1 mmol) was added to thesolution. The resulting mixture was stirred at 0° C. for 15 minutes andethidium bromide (EB) (200 mg, 0.5 mmol) was then added. The resultingmixture was stirred at 0° C. for one hour and then at room temperatureovernight. The resulting solution was concentrated to dryness in vacuoand the resulting residue was purified by column chromatography onsilica gel to give a dark red solid (155 mg), Substrate No. 5, asgenerally represented by the structure and legend below, and anotherdark red solid (70 mg), Substrate No. 6, as generally represented by thestructure and legend below, namely, (Z-Ala-Ala)₂-EB (70 mg). Each ofSubstrate No. 5 and Substrate No. 6, and information concerning same,may be found in Table 2.

Example 27 Preparation of Compound No. 24

Compound No. 24 (56 mg), as generally represented by the structurebelow, was prepared from Compound No. 16 (128.3 mg) using the generalprocedure described above in relation to Example 7.

Example 28 Preparation of Compound No. 25

A suspension of sodium hydride (60% weight purity) (101 mg, 2.52 mmol)in DMF (5 mL) at 0° C. was prepared. 2-methyl-5,6-dichlorobenzimidazole(507.6 mg, 2.52 mmol) was added to the suspension in one portion. Theresulting mixture was stirred at 0° C. for 30 minutes and benzylchloride (290 μL, 2.52 mmol) was added. The resulting mixture was thenstirred at room temperature overnight and concentrated to dryness invacuo. The resulting residue was purified by column chromatography onsilica gel using CHCl₃:EtOAc:hexanes=3:3:4 as the solvent to give anoff-white solid (500 mg), Compound No. 25, as generally represented bythe structure below.

Example 29 Preparation of Compound No. 26

A mixture of Compound No. 25, namely,1-benzyl-2-methyl-5,6-dichlorobenzimidazole (250 mg, 0.86 mmol), and4-(bromomethyl)benzoic acid (500 mg, 2.15 mmol) in chlorobenzene (5 mL)was heated at 120° C. overnight. The resulting mixture was allowed tocool to room temperature, EtOAc (20 mL) was added, and the resultingsuspension was refluxed gently for 2 hours. Compound No. 26 (326 mg), asgenerally represented by the structure below, was collected from therefluxed suspension by suction filtration.

Example 30 Preparation of Compound No. 27

A solution of Compound No. 26 (200 mg, 0.32 mmol) and2-(2-anilinovinyl)-1-methylbenzoxazolium iodide (150 mg, 0.59 mmol)(prepared from 2-methylbenzoxazole using the general procedure describedabove in relation to Example 2) in DMF (3 mL) was prepared. Aceticanhydride (110 μL, 1.20 mmol) and Et₃N (250 μL, 1.71 mmol) were added tothe solution. The resulting solution was stirred at room temperature for7 hours and EtOAc (5 mL) was added. The resulting suspension was stirredat room temperature overnight and a solid product (200 mg), Compound No.27, as generally represented by the structure below, was collected fromthe suspension by suction filtration. Compound No. 27 is an example of amembrane dye comprising a carboxylic acid functional group.

Example 31 Preparation of Compound No. 28

A solution of Compound No. 27 (100 mg, 0.15 mol) in DMF (2 mL) wasprepared. Et₃N (25 μL, 0.18 mmol) and TSTU (45 mg, 0.15 mmol) were addedto the solution. The resulting mixture was stirred at room temperaturefor 30 minutes and Et₃N (75 μL) and tert-butyl carbazate (29 mg, 0.18mmol) were added. The mixture was stirred at room temperature overnightand then poured into a solution of NaI (0.5 g) in water (50 mL). Aprecipitate was collected from the resulting solution and then purifiedon silica gel using MeOH/CHCl₃. The purified compound was dissolved inCH₂Cl₂ (5 mL) and stirred at 5° C. TFA (1 mL) was added to the cooledsolution. The resulting mixture was stirred at 5° C. for 2 hours andthen concentrated to dryness in vacuo. The resulting product, CompoundNo. 28, as generally represented by the structure below, was used toprepare Compound No. 29, as described below in relation to Example 32,without further purification. Compound No. 28 is an example of amitochondrial dye comprising a hydrazide functional group.

Example 32 Preparation of Compound No. 29

A solution of Fmoc-Asp(OtBu)-OH (77 mg, 0.182 mmol) in DMF (5 mL) atroom temperature was prepared. Et₃N (30 μL, 0.21 mmol) and TSTU (55 mg,0.185 mmol) were added to the solution. The resulting mixture wasstirred at room temperature for 1 hour Et₃N (25 μL) and Compound No. 28(75 mg, 0.091 mmol) were added successively. The resulting mixture wasstirred at room temperature overnight and then poured into a solution ofNaI (0.5 g) in water (about 50 mL). A precipitate was collected from theresulting solution and then purified by column chromatography on silicagel to give an orange solid (95 mg), Compound No. 29, as generallyrepresented by the structure below.

Example 33 Preparation of Substrate No. 47

A solution of Compound No. 29 (75 mg) and piperidine (0.5 mL) in CHCl₃(3 mL) was stirred at room temperature for 3 hours. The resultingsolution was concentrated to dryness in vacuo. The resulting residue wasredissolved in CH₂Cl₂ (5 mL) and TFA (1 mL) was added to the resultingsolution. The resulting mixture was stirred at room temperatureovernight and then concentrated to dryness in vacuo. The resultingresidue was triturated with EtOAc to give an orange solid enzymesubstrate (45 mg), Substrate No. 47, as generally represented by thestructure below. Substrate No. 47 and information concerning same may befound in Table 6.

Example 34 Preparation of Compound No. 30

5,6-Dichloro-2-methylbenzimidazole (5 g), 3-bromopropanol (6equivalents) and K₂CO₃ (10 equivalents) were mixed in DMF (50 mL). Theresulting mixture was stirred at 120° C. overnight and then poured intoa saturated NaCl solution (about 200 mL). A solid product, Compound No.30, as generally represented by the structure below, was collected fromthe resulting solution and dried under vacuum.

Example 35 Preparation of Compound No. 31

Compound No. 31, as generally represented by the structure below, wasprepared from 5,6-dichloro-2-methylbenzimidazole and ethyl iodide usingthe general procedure described above in relation to Example 34.

Example 36 Preparation of Compound No. 32

Compound No. 32, as generally represented by the structure below, wasprepared from N,N′-diphenylformamidine (10 equivalents) and Compound No.31 (1 equivalent) following the general procedure described above inrelation to Example 2.

Example 37 Preparation of Compound No. 33

Compound No. 32 (1 g) and Compound No. 30 (0.7 g) were coupled usingNEt₃/Ac₂O via the general procedure described above in relation toExample 30. At the end of the coupling reaction, a solution of NaOH (0.5g) in water (5 mL) was added to the resulting mixture and allowed toreact for half an hour to hydrolyze any acetate form of the dye. Theresulting solution was then poured into a saturated NaCl solution (100mL). A precipitate was collected from the resulting solution and thendried under high vacuum at 50° C. for at least 24 hours to give CompoundNo. 33, as generally represented by the structure below.

Example 38 Preparation of Compound No. 34

A mixture of Compound No. 33 (2 g, 3.2 mmoles), Hg(CN)₂ (2.97 g, 10.7mmoles) and 4 Å molecular sieves (20 g) in dry CH₃CN (70 mL) was stirredunder nitrogen for one hour. 2,3,4-Tri-O-acetyl a-D-glucopyano-siduronicacid methyl ester (2.8 g, 7.04 mmoles) was added portion-wise to theresulting solution. The resulting mixture was stirred until TLCindicated complete reaction. The resulting mixture was suction filteredthrough a Celite pad to remove any precipitate. The filtrate wasevaporated and the crude product, Compound No. 34, as generallyrepresented by the structure below, was purified on a silica columneluted with MeOH/CHCl₃.

Example 39 Preparation of Compound No. 35

Compound No. 34 was deprotected using NaOMe and LiOH successivelyaccording to the general procedure described in U.S. Pat. No. 5,208,148to give Compound No. 35, as generally represented by the structurebelow. Compound No. 35 is an example of an enzyme substrate for aβ-glucuronidase enzyme.

Example 40 Preparation of Compound No. 36

Compound No. 36, as generally represented by the structure below, wasprepared from lepidine and 1,2-dibromoethane (20 equivalents) via thegeneral procedure described above in relation to Example 3.

Example 41 Preparation of Compound No. 37

Compound No. 37, as generally represented by the structure below, wasprepared from Compound No. 5 and Compound No. 36 via the generalprocedure described above in relation to Example 6.

Example 42 Preparation of Compound No. 38

Compound No. 38, as generally represented by the structure below, wasprepared from Compound No. 37 via treatment with KSCSOEt and then withAcNHNH₂ as described for thiol compounds in U.S. Pat. No. 5,955,604.

Example 43 Preparation of Compound No. 39

Compound No. 39, as generally represented by the structure below, wasprepared from ACLH (Otsuka Chemical Co., Ltd., Osaka, Japan) andglutaric anhydride in the presence of triethylamine via a proceduredescribed by Gao et al., J. Am. Chem. Soc. 125, 11146 (2003) for makingsimilar compounds.

Example 44 Preparation of Compound No. 40

Compound No. 40, as generally represented by the structure below, wasprepared by coupling Compound No. 38 and Compound No. 39 using aprocedure similar to that described in U.S. Pat. No. 5,955,604.

Example 45 Preparation of Compound No. 41

Compound No. 41, as generally represented by the structure below, wasprepared by deprotection of Compound No. 40 using TFA/anisole asgenerally described by Gao et al., J. Am. Chem. Soc. 125, 11146 (2003).Compound No. 41 is an example of nucleic acid dye-based enzyme substratefor a β-lactamase enzyme.

Example 46 Preparation of Compound No. 42

A mixture of picoline (10 g, 0.11 mol) and 4-bromobutyric acid (25 g,0.15 mol) was heated at 120° C. for 5 hours. After the resulting mixturewas cooled to room temperature, EtOAc (100 mL) was added and theresulting mixture was refluxed gently for 1 hour. Compound No. 42, asgenerally represented by the structure below, was collected from therefluxed mixture by suction filtration.

Example 47 Preparation of Compound No. 43

A mixture of 4-N,N-diethylaminobenzaldehyde (1.5 g, 8.46 mmol), CompoundNo. 42 (2.2 g, 8.46 mmol) and piperidine (0.1 mL, 1 mmol) in CH₃OH (20mL) was stirred at reflux temperature overnight. The resulting solutionwas concentrated to dryness in vacuo and the resulting residue waspurified by column chromatography on silica gel to give a dark red solid(3 g), Compound No. 43, as generally represented by the structure below.

Example 48 Preparation of Compound No. 44

Compound No. 44, as generally represented by the structure below, wasprepared by first coupling Compound No. 43 and Compound No. 15 accordingto the general procedure described above in relation to Example 18, andthen deprotecting the resulting conjugate with TFA according to thegeneral procedure described above in relation to Example 19. CompoundNo. 44 is an example of a membrane dye-based enzyme substrate.

Example 49 Preparation of Compound No. 45

Compound No. 45, as generally represented by the structure below, may beprepared by coupling Compound No. 43 and the synthetic peptidePro-Asn-Gly-Leu-Glu-Ala-D-Arg-D-Arg-D-Arg-NH₂ (custom-synthesized by GLBiochem (Shanghai) Ltd., Shanghai, China), or PQGLEA-D-R-D-R-D-R—NH₂,using the general procedure described in relation to Example 18. Theresulting conjugate may be purified by preparative HPLC. Compound 45 isan example of a membrane dye-based enzyme substrate.

Example 50 Preparation of Compound No. 46

Compound No. 46, as generally represented by the structure below, wasprepared from Compound No. 43 and mono-t-Boc-ethylenediamine accordingto the general procedure described above in relation to Example 7.

Example 51 Measurement of Fluorescence Spectra in the Presence of DNA

Stock solutions of nucleic acid dye-based substrates and respectivecontrol compounds were prepared by dissolving each compound in DMSO at 1mg/mL concentration. The stock solutions were diluted into a pH 7.4buffer (TE buffer) comprising Tris (10 mM), EDTA (1 mM) and NaCl (50 mM)to give a final concentration of 1 μM. Dye solutions containing anexcess amount of DNA were prepared by adding a sufficient amount of calfthymus DNA calculated to yield a ratio of greater to or equal to 50 DNAbase pairs per dye molecule. A fluorescence spectrum for each dye wasrecorded before and after the addition of DNA using a Jasco F-750fluorescence spectrophotometer, with the fluorescence excitationwavelength set at λ_(A)-25 nm or 600 nm, where λ_(A) is the absorptionmaximum for each dye in the presence or the absence of DNA, and emissioncollection wavelength set at 660 nm. The emission spectra associatedwith Substrate No. 20 and control Compound No. 24 are shown in FIG. 1.The data shows that the control Compound No. 24, the enzymaticallycleaved product of Substrate No. 20, is substantially more fluorescentthan Substrate No. 20 itself.

Example 52 DNA Titration of Nucleic Acid Dye-Based Substrates andControls

In this example, nucleic acid dye-based caspase-3 substrates, SubstrateNo. 19 and Substrate No. 20, and respective control compounds, CompoundNo. 7 and Compound No. 24, were titrated with varying amounts of calfthymus dsDNA and the fluorescence signals of the solutions wererecorded. Briefly, the substrates and the respective controls wereprepared as generally described above in relation to Example 51.Multiple wells of a 96-well plate were loaded with the substrate (100μL, 1 μM) or the respective control (100 μL, 1 μM). Each well was thentitrated with 5 μL of one of the seven DNA stock solutions of 2.1, 4.1,8.2, 16.4, 32.8 65.6 and 127.5 μg/mL concentration, respectively, in pH7.4 TE buffer. After a 30-minute incubation at room temperature, thefluorescence reading of each well was recorded with a SpectraMax GerminiXS fluorescence microplate reader (Molecular Devices Corp., Sunnyvale,Calif.), with the excitation wavelength and the emission collectionwavelength set at 485 nm and 530 nm, respectively. The titration curvesfor each pair of substrate and control dyes are shown in FIG. 2. Thedata shows that the substrates are relatively insensitive to thepresence of DNA over a wide DNA concentration range, while the controls,the enzymatically cleaved products of the substrates, are fluorescentlyresponsive to the amount of DNA present.

Example 53 In Vitro Enzymatic Assay for Nucleic Acid Dye-BasedSubstrates for Caspase-3

In this example, nucleic acid dye-based caspase-3 substrates wereassayed in the presence of caspase-3 and dsDNA by monitoring thefluorescence increase. In one example, Substrate No. 19 (10 μM) wasincubated with caspase-3 (0.1 unit/mL) (Biovision Inc., Mountain View,Calif.) and salmon sperm dsDNA (3.3 μg/mL) in a caspase assay buffer(100 μL; HEPES buffer (50 mM, pH 7.4), NaCl (100 mM), EDTA (1 mM), CHAPS(0.1%), DTT (10 mM), PMSF (1 mM) and glycerol (10%)) in a well of ablack 96-well plate. The plate was then placed in a SpectraMax GerminiXS fluorescence microplate reader (Molecular Devices Corp., Sunnyvale,Calif.), with the excitation wavelength set at 485 nm and emissioncollection wavelength set at 530 nm, to measure the fluorescence signalover time, as shown in FIG. 3. The data demonstrates that Substrate No.19 is a substrate for caspase-3 enzyme.

Example 54 Detection of Caspase-3 Activity in Live Cells by FlowCytometry

In this example, Jurkat cells were used to test whether Substrate No. 19may be used to detect caspase-3 activity, or a lack thereof, within livecells by generating a nucleic acid dye that forms fluorescence uponbinding to DNA. A flask of Jurkat cells were induced with staurosporine(1 μM) for apoptosis. Aliquots of the induced Jurkat cells were taken at1 hour, 2.5 hours and 5 hours after induction, respectively, andaliquots of uninduced Jurkat cells, which served as negative controls,were taken at the same times. Substrate No. 19 was added to each Jurkatcell culture medium to give a final concentration of 10 μM and eachresulting medium was allowed to incubate for 15 minutes beforeundergoing flow cytometry analysis using the FL1 channel for greenfluorescence. The results, shown in FIG. 4, show that the amount ofstaurosporine stimulation time strongly correlates with the percentageof caspase-3-positive cells identified by Substrate No. 19. For example,a stimulation time of 1 hour, 2.5 hours and 5 hours was associated witha 10%, 80% and 97% caspase-3-positive cell identification, respectively,as may be seen in the representations B, C and D of FIG. 4,respectively. One hour of staurosporine induction was required forSubstrate No. 19 to reliably detect the presence of caspase-3-positivecells. Detection of intracellular caspase-3 activity via Substrate No.19 is possible after only 15 minutes of incubation time, withoutnecessitating cell destruction. Thus, it may be possible to continuouslymonitor enzymatic activity over more or less the entire course ofcellular life via an enzyme substrate such as Substrate No. 19.

In this example, Jurkat cells were similarly used to evaluate DEVD-R110(Biotium Inc., Hayward, Calif.), where DEVD is associated with SEQ IDNO: 1, a sensitive fluorogenic caspase-3 substrate that detectscaspase-3 only after cell lysis. Aliquots of induced and uninducedJurkat cells, were taken at the same intervals described above, lysedand then incubated with DEVD-R1110 (where DEVD is associated with SEQ IDNO: 1) to confirm the presence or the absence of caspase-3 activity. Twohours of staurosporine induction were required for DEVD-R110 (where DEVDis associated with SEQ ID NO: 1) to detect the presence ofcaspase-3-positive cells (data not shown). Detection of intracellularcaspase-3 activity via DEVD-R110 (where DEVD is associated with SEQ IDNO: 1) calls for cell destruction of the cells in order to access theenzyme residing within the cells. It may be possible to obtain a snapshot of intracellular enzyme activity via an enzyme substrate such asDEVD-R110 (where DEVD is associated with SEQ ID NO: 1). The differencesbetween the results associated with Substrate No. 19 and DEVD-R110(where DEVD is associated with SEQ ID NO: 1) suggest that the former mayhave some advantages relative to the latter.

Example 55 Detection of Caspase-3 Activity in Live Cells by FluorescenceMicroscopy

Substrate No. 19 (10 μM) was incubated for 15 minutes with Jurkat cellsthat had been induced for 4 hours with staurosporine (1 μM). Separately,Substrate No. 19 (10 μM) was incubated for 15 minutes with uninducedJurkat cells, which served as a negative control. For each of the cellcultures, cells (1×10⁵) were taken, pelleted and then resuspended inAnnexin V binding buffer (100 μL; HEPES buffer (10 mM, pH 7.4), NaCl(140 mM) and CaCl₂ (2.5 mM)) in a tube, whereupon Texas Red-conjugatedAnnexin V (5 μL, 50 μg/mL) (Biotium Inc., Hayward, Calif.) was added toeach tube. After 15 minutes of incubation at room temperature, a cellculture (5 μL) of the induced cells or the uninduced cells was spottedonto a slide, which was then mounted with a coverslip and sealed withnail polish. The cells were examined with a 510 Meta UV/Vis confocalmicroscope. Distinctive populations of fluorescently-labeled cells wereobserved, as may be seen in the images of FIG. 5, as follows: cells withonly green fluorescence in cellular nuclei; cells with only redfluorescence on a cytoplasmic membrane; and cells with greenfluorescence in cellular nuclei and red fluorescence on a cytoplasmicmembrane. The green fluorescence is indicative of nuclear DNA stainingby the nucleic acid dye that is formed from substrate cleavage viacaspase-3. The red fluorescence is indicative of the staining ofphosphatidylserine (PS) by the Texas Red-conjugated Annexin V, afluorescent stain that is used to identify apoptotic cells. The stainingpattern shows that the amount of PS present on the cell membranes andthe amount of caspase-3 activity are not necessarily correlated, as isindicative of a heterogeneous population of apoptotic cells.

Example 56 DNA Titration of Compound No. 21 and Compound No. 23

DNA titrations of Compound No. 21, a nucleic acid dye-based HDACsubstrate, and Compound No. 23, a control associated with Compound No.21, were titrated with varying amounts of calf thymus dsDNA in themanner generally described above in relation to Example 52. Thetitration curves for substrate and the control are shown in FIG. 6. Thedata shows that the substrates are relatively insensitive to thepresence of DNA over a wide DNA concentration range, while the controls,the enzymatically cleaved products of the substrates, are fluorescentlyresponsive to the amount of DNA present.

Example 57 Liposome Titrations of Compound No. 44 and Compound No. 46

A stock solution (5 mM) of Compound No. 44, a membrane dye-basedcaspase-3 substrate, was prepared by dissolving Compound No. 44 indeionized water. A stock solution (5 mM) of Compound No. 46, theenzymatic cleavage product of Compound No. 44, was prepared bydissolving Compound No. 46 in Di-H₂O. A liposome stock solution wasprepared by suspending 1,2-dioleyl-sn-glycero-3-phosphocholine (DOGPC)(Avanti Polar Lipids, Inc., Alabaster, Ala.) in a liposome buffer (2.5mg/mL; pH 7.5; HEPES buffer (10 mM), NaCl (150 mM), CaCl₂ (2 mM) andMgCl₂ (2 mM)) and then sonicating the resulting suspension for 30minutes. A solution of either Compound No. 44 or the control CompoundNo. 46 in the liposome buffer was prepared and the resulting solution(50 μM) was titrated with varying amounts of the liposome stocksolution. Fluorescence signals associated with various such solutionswere recorded on a SpectraMax Germini XS fluorescence microplate reader(Molecular Devices Corp., Sunnyvale, Calif.). Fluorescence readingsassociated with the substrate, Compound No. 44, or the control, CompoundNo. 46, were plotted in relation to the concentration of liposomes, asmay be seen in FIG. 7. The membrane dye-based substrate, Compound No.44, appeared to be relatively unresponsive to the change in liposomeconcentration, while the control, Compound No. 46, the enzymaticcleavage product of the substrate, appeared to be responsive to thechange in liposome concentration, with the fluorescence intensityincreasing as the liposome concentration increased. The data indicatesthat the substrate is nonfunctional as a membrane dye, while theenzymatically cleaved product of the substrate is a fully functionalmembrane dye that becomes fluorescent upon partitioning into membranes.

An enzyme substrate of the invention may be used to detect the presenceof an enzyme via fluorescence. FIGS. 8 and 9 provide schematicillustrations of examples of such use of enzyme substrates, which employvarious biomolecules. As shown, an enzyme may be used to interact withan enzyme substrate to produce a functional dye, and a partner molecule,partner biomolecules, and/or an assembly of partner molecules may beused to interact with that functional dye to produce fluorescence. Thepresence or absence of fluorescence may be used as an indicator ofenzyme presence or absence, respectively. Fluorescence detection maythus be employed to evaluate enzymatic activity.

Merely by way of example, an enzyme, such as caspase-3, for example, maybe used to cleave a nucleic acid dye-based capase-3 substrate of theinvention, as schematically illustrated in FIG. 8. The cleavage mayresult in a relatively or fully functional nucleic acid-binding dye thatmay be fluorogenic or nonfluorescent. A partner molecule, such as a DNAmolecule, for example, may be used to bind to the enzymatically releasednucleic acid-binding dye to form a complex of sufficiently detectable tohigh fluorescence. Any fluorescence generated by the complex may then bedetected. Further, merely by way of example, an enzyme, such ascaspase-3, for example, may be used to cleave a membrane-based caspase-3substrate of the invention, as schematically illustrated in FIG. 9. Thecleavage may result in a relatively or fully functional membrane dyethat may be fluorogenic or nonfluorescent. A partner molecule or partnermolecules, such as an assembly of lipid molecules, for example, may beused to enable the enzymatically released membrane dye to partition intothe assembly. Merely by way of example, the assembly of lipid moleculesmay be a membrane and the partition of the dye may be into the membrane,as schematically illustrated in FIG. 9. Upon this partition,fluorescence may be generated and detected.

An enzyme substrate of the invention may have at least two capabilitiesor functionalities, one being the detection of an enzyme and anotherbeing the staining of a biomolecule or biomolecules. Such an enzymesubstrate may be referred to herein as a biofunctional enzyme substrate.A bifunctional enzyme substrate of the invention may be useful in thedetection of intracellular enzyme activity in live cells. This detectionmay be of interest because the presence or absence of enzyme activitymay be accompanied by other biological change(s) within cells. An enzymesubstrate of the invention may thus be useful in the detection of theenzyme activity as well as other biological event(s) within cells, suchas a simultaneous biological event, for example.

Merely by way of example, an enzyme substrate of the invention may beuseful in the study of apoptosis. Apoptosis, also called programmed celldeath, is a normal physiological process that occurs in the developmentof embryos and in the maintenance of tissue homeostasis. Improperlyregulated apoptosis can lead to several pathological conditions,including cancer and neurodegenerative diseases. (Reed et al., Curr.Opin. Biotechnol. 11, 586 (2000); Wellington et al., Clin. Genet. 57, 1(2000); and Loww et al., Carcinogenesis 21, 485 (2000)). Apoptosis isregulated via the activation of enzymes called caspases, one of which isthe caspase-3 enzyme shown in FIGS. 8 and 9. Apoptotic cells may becharacterized by caspase activation and other intracellular event(s),such as the condensation of chromatin, the cleavage of nuclear DNA,nuclear blebbing, the shrinking of cell cytoplasm and organelles, andthe loss of mitochondrial membrane potential, for example. A nucleicacid dye-based caspase substrate of the invention may be useful todetect caspase activity and to monitor change in the cell nucleus, asmay be characteristic of apoptotic cells. (See Example 10, Substrate No.19, and FIG. 5, for example.) A cytoplasmic membrane-based caspasesubstrate of the invention may be useful to detect the caspase activityand to monitor of cytoplasm shrinkage, as may be characteristic ofapoptotic cells. (See Example 33, Substrate No. 47.)

An enzyme substrate of the invention may have a variety of other usefulproperties and uses. Merely by way of example, an enzyme substrate maybe such that a fluorescent signal generated in response to enzymeactivity associated with the substrate may be retained well within acell. For example, the enzymatic product of the substrate mayfluorescently and actively bind to an internal component of the cell.The retention of a fluorescence signal in this manner facilitates theintracellular detection of enzyme activity in live cell studies. Livecell studies may be of interest (relative to studies based on celllysates, for example) because they permit continuous monitoring of thecells of interest and preserve the integrity of the overall cellularfunctions.

An enzyme substrate of the invention may comprise any of a variety offunctional dyes having excitation and emission wavelengths that span theentire visible spectrum and extend into the near infrared spectralregion. (See the wavelengths shown in Tables 2, 3, 4, 5, 6 and 7, forexample.) One or more enzyme substrate(s) having different fluorescencewavelengths may be used to detect multiple enzyme activities within thesame cell. One or more enzyme substrate(s) having different fluorescenceemission wavelengths may be combined with one or more non-enzymesubstrate fluorescent probe(s) having still different fluorescenceemission wavelengths to image or to detect multiple cellular targets atthe same time.

At least some of the enzyme substrates of the invention may beadvantageous by virtue of their relatively long excitation and emissionwavelengths. Merely by way of example, a biological sample and/or avessel containing a biological sample may emit blue fluorescence whenexcited by a UV light. A fluorescent dye with an excitation wavelengthand an emission wavelength of about 450 nm or more may be useful inbiological imaging associated with such a sample or vessel to reduce orto minimize background fluorescence from the sample or the vessel, forexample. Merely by way of example, in a biological application, afluorescent dye having an excitation wavelength and an emissionwavelength of about 470 nm or more may be usefully employed to reduce orto minimize background fluorescence. In general, an enzyme substrate ofthe invention may comprise a functional dye having an excitationwavelength and an emission wavelength of about 480 nm or more, asdemonstrated by the information in Tables 2, 3, 4, 5, 6 and 7. Forexample, Substrate No. 19 of Example 10 comprises a functional dye thathas an excitation wavelength of about 515 nm and emission wavelength ofabout 530 nm. It will be understood that an enzyme substrate comprisinga functional dye associated with shorter wavelengths, such as anexcitation wavelength and an emission wavelength of about 450 nm or lessmay be useful in any of a variety of applications, such as in amulticolor imaging experiment in which the blue fluorescence of thesubstrate is one of several colors useful or necessary in theexperiment, merely by way of example. In general, an enzyme substratecomprising a functional dye having an excitation wavelength and anemission wavelength from about 350 nm to about 450 nm, or longer, asdescribed above, is contemplated as being within the scope of thepresent invention.

At least some of the enzyme substrates of the invention may beadvantageous in that they require only a single enzymatic cleavage togenerate a functional dye. For example, with respect to each ofSubstrate No. 19 of Example 10, Substrate No. 20 of Example 19, andSubstrate No. 5 of Example 26, a single enzymatic cleavage is sufficientto release a nucleic acid dye. Further by way of example, with respectto each of Substrate No. 47 of Example 33 and Compound No. 44 of Example48, a single enzymatic cleavage is sufficient to release a membrane dye.An enzyme substrate that may be cleaved to release a functional dye viaa single cleavage may accelerate the rate of enzyme detection andsimplify enzyme kinetics for any quantitative analysis.

The present invention provides a number of useful enzyme substrates,such as any one or more of the following enzyme substrates: caspase,matrix metalloprotease (MMP), collagenase, gelatinase, β-lactamase,phosphatase, glycosidase, g-secretase, HCV protease, cathepsin (such ascathepsin B or cathepsin L, for example), trypsin, chymotrypsin, HIVprotease, elastase, rennin protease, and phosphatidylinositol-specificphospholipase C, merely by way of example. The present inventionprovides a nucleic acid dye-based enzyme substrate that is suitable forintracellular detection of caspase-3 in live cells, merely by way ofexample. The present invention also provides a method for preparing anyof the foregoing enzyme substrates and a method of using any of theforegoing substrates. Any method of using a composition of the presentinvention is contemplated as part of the present invention. The presentinvention further provides any kit comprising an enzyme substrate thatmay be useful. Merely by way of example, such a kit for determiningapoptosis may comprise an enzyme substrate of the present invention andany other suitable, desirable or necessary component or components, suchas any component useful for detection, or such as any useful detectiondevice. Further, merely by way of example, the present inventionprovides a kit for detecting presence, absence, or activity of an enzymewhich may comprise an inhibitor of activity of the enzyme, optionally, apromoter of activity of the enzyme, and an enzyme substrate for theenzyme, as well as a kit for determining an effect of a substance on anenzyme which may comprise an inhibitor of activity of the enzyme and anenzyme substrate for the enzyme. Any kit comprising an enzyme substrateof the present invention that has useful application is contemplated aspart of the present invention.

Enzyme substrates and associated technology, including associatedsystems, kits, methods, and the like, of the present invention areprovided. An enzyme substrate of the invention may comprise abiologically functional fluorescent dye and an enzyme-specific substratemoiety attached in such a way that the functionality of the functionaldye is diminished. An enzymatic reaction may cleave at least a portionof the substrate moiety from the enzyme substrate to provide a morefunctional product dye. This product dye may be nonfluorescent or weaklyfluorescent, in general, and relatively fluorescent, in a particularcondition, such as when bound to a partner molecule, partner molecules,or an assembly of partner molecules. An enzyme substrate of the presentinvention may thus be useful in fluorescence detection, and/or in any ofa variety of useful applications, such as the detection of enzymaticactivity in a cell-free system or in a living cell, the screening ofdrugs, or the diagnosis of disease.

Various modifications, processes, as well as numerous structures towhich the present invention may be applicable will be readily apparentto those of skill in the art to which the present invention is directed,upon review of the specification. Various references, publications,provisional and non-provisional United States or foreign patentapplications, and/or United States or foreign patents, have beenidentified herein, each of which is incorporated herein in its entiretyby this reference. Various aspects and features of the present inventionhave been explained or described in relation to understandings, beliefs,theories, underlying assumptions, and/or working or prophetic examples,although it will be understood that the invention is not bound to anyparticular understanding, belief, theory, underlying assumption, and/orworking or prophetic example. Although the various aspects and featuresof the present invention have been described with respect to variousembodiments and specific examples herein, it will be understood that theinvention is entitled to protection within the full scope of theappended claims.

1. A fluorogenic enzyme substrate of formula:DYE-(B)_(m) wherein: DYE comprises a fluorogenic dye which is anasymmetric cyanine dye of formula:

wherein: X comprises O or S; n is selected from 0, 1, 2 and 3; ψcomprises a biologically compatible counter ion; d is a number of ψsufficient to render overall charge of the substrate neutral; the dottedline represents atoms of at least one fused aromatic ring, optionallycomprising at least one quaternized or unquaternized nitrogen; at leastone ligand, but no more than two ligands, of ligands R₄, R₅, R₆ and R₇comprises at least one B which is an enzyme substrate moiety capable ofreacting with said enzyme, such that said reacting results in anincrease in the fluorescence of said fluorogenic dye; when ligand R₄does not comprise B, ligand R₄ comprises H; a C1 to about C6 alkyl; a C1to about C6 alkoxy; a halogen; or an aryl meta to X, wherein the aryloptionally comprises at least one hetero atom of hetero atoms N, O andS; when ligand R₅ does not comprise B, ligand R₅ comprises a C1 to aboutC6 alkyl; when ligand R₆ does not comprise B, ligand R₆ comprises H; aC1 to about C10 alkyl, wherein the alkyl optionally comprises at leastone hetero atom of hetero atoms N, O and S; a halogen; a C1 to C10alkoxy, wherein the alkoxy optionally comprises at least one hetero atomof hetero atoms N, O, and S; a C1 to C10 alkylmercapto, wherein thealkylmercapto comprises at least one hetero atom of hetero atoms N, O,and S; a C2 to about C12 dialkylamino, wherein the dalkylaminooptionally comprises at least one hetero atom of hetero atoms N, O, andS; a substituted or an unsubstituted aryl, wherein the aryl optionallycomprises 1 to 3 hetero atom(s) of hetero atoms N, O, S and a halogen;when ligand R₇ does not comprise B, ligand R₇ comprises H; a C1 to aboutC10 alkyl, optionally comprising an aryl, and optionally comprising atleast one hetero atom of hetero atoms N, O, and S; a substituted or anunsubstituted aryl, optionally containing 1 to 3 hetero atom(s) ofhetero atoms N, O, S and a halogen; and independently, each ligand ofligands R₈ and R₉ comprises H; a C1 to about C10 alkyl, optionallycomprising at least one hetero atom of hetero atoms N, O, and S; ahalogen; a C1 to C10 alkoxy, wherein the alkoxy optionally comprises atleast one hetero atom of hetero atoms N, O, and S; a C1 to C10alkylmercapto, wherein the alkylmercapto optionally comprises at leastone hetero atom of hetero atoms N, O, and S; a C2 to about C12dialkylamino, optionally comprising at least one hetero atom of heteroatoms N, O, and S; or a substituted or an unsubstituted aryl, optionallycomprising 1 to 3 hetero atom(s) of hetero atoms halogen, N, O and S; orligand R₈ and ligand R₉ in combination, form a fused aromatic ring,wherein the ring is optionally substituted by at least one substituentof a C1-C2 alkyl substituent, a C1-C2 alkoxy substituent, a C1-C2alkyl-mercapto substituent, and a halogen substituent.
 2. The substrateof claim 1, wherein the dye comprises an asymmetric cyanine dye having astructural formula,

wherein: the dotted line represents atoms of at least one fused aromaticring, optionally comprising at least one quaternized or unquaternizednitrogen; X comprises O or S; n is selected from 0, 1, 2 and 3; ψcomprises a cation selected from H+, Na+, K+, NH₄+,N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anionselected from a halide, a sulfate, a phosphate, a perchlorate, ahexafluorophosphate, a trifluoroacetate, and a tetrafluoroborate; d is anumber of ψ sufficient to render overall charge of the substrateneutral; at least one ligand, but no more than two ligands, of ligandsR₄, R₅, R₆ and R₇ has a structural formula, -L-W—B, wherein: L comprisesan aliphatic C2 to about C10 linker, optionally comprising an aryl, andoptionally comprising at least one hetero atom of hetero atoms O, S, N,F, and Cl; W comprises at least one atom selected from atoms O, N and S;and if B comprises an amino acid or a peptide, B is attached to W via aC-linkage; when ligand R₄ does not comprise -L-W—B, ligand R₄ comprisesH; a C1 to about C6 alkyl; a C1 to about C6 alkoxy; a halogen; or anaryl meta to X, wherein the aryl optionally comprises at least onehetero atom of hetero atoms N, O and S; when ligand R₅ does not comprise-L-W—B, ligand R₅ comprises a C1 to about C6 alkyl; when ligand R₆ doesnot comprise -L-W—B, ligand R₆ comprises H; a C1 to about C10 alkyl,wherein the alkyl optionally comprises at least one hetero atom ofhetero atoms N, O and S; a halogen; a C1 to C10 alkoxy, wherein thealkoxy optionally comprises at least one hetero atom of hetero atoms N,O, and S; a C1 to C10 alkylmercapto, wherein the alkylmercapto comprisesat least one hetero atom of hetero atoms N, O, and S; a C2 to about C12dialkylamino, wherein the dalkylamino optionally comprises at least onehetero atom of hetero atoms N, O, and S; a substituted or anunsubstituted aryl, wherein the aryl optionally comprises 1 to 3 heteroatom(s) of hetero atoms N, O, S and a halogen; when ligand R₇ does notcomprise -L-W—B, ligand R₇ comprises H; a C1 to about C10 alkyl,optionally comprising an aryl, and optionally comprising at least onehetero atom of hetero atoms N, O, and S; a substituted or anunsubstituted aryl, optionally containing 1 to 3 hetero atom(s) ofhetero atoms N, O, S and a halogen; and independently, each ligand ofligands R₈ and R₉ comprises H; a C1 to about C10 alkyl, optionallycomprising at least one hetero atom of hetero atoms N, O, and S; ahalogen; a C1 to C10 alkoxy, wherein the alkoxy optionally comprises atleast one hetero atom of hetero atoms N, O, and S; a C1 to C10alkylmercapto, wherein the alkylmercapto optionally comprises at leastone hetero atom of hetero atoms N, O, and S; a C2 to about C12dialkylamino, optionally comprising at least one hetero atom of heteroatoms N, O, and S; or a substituted or an unsubstituted aryl, optionallycomprising 1 to 3 hetero atom(s) of hetero atoms halogen, N, O and S; orligand R₈ and ligand R₉, in combination, form a fused aromatic ring,wherein the ring is optionally substituted by at least one substituentof a C1-C2 alkyl substituent, a C1-C2 alkoxy substituent, a C1-C2alkyl-mercapto substituent, and a halogen substituent.
 3. The substrateof claim 2, wherein: when ligand R₄ does not comprise -L-W—B, ligand R₄comprises H; a methoxy meta to X; or an aryl meta to X, wherein the aryloptionally comprises at least one hetero atom of hetero atoms N, O andS; when ligand R₅ does not comprise -L-W—B, ligand R₅ comprises amethyl; when ligand R₆ does not comprise -L-W—B, ligand R₆ comprises H;a C1 to about C6 alkyl; a C1 to about C6 alkoxy; a C1 to about C6alkylmercapto; a C2 to about C12 dialkylamino, optionally comprising oneN; or a substituted or an unsubstituted aryl, optionally comprising 1 to3 hetero atom(s) of hetero atoms N, O and S; when ligand R₇ does notcomprise -L-W—B, ligand R₇ comprises a C1 to about C3 alkyl, optionallycomprising an aryl; and each ligand of ligands R₈ and R₉ comprises H; orligand R₈ and ligand R₉, in combination, form a fused 6-membered ring,wherein the ring is optionally substituted by at least one substituentof a methyl substituent, a methoxy substituent, a methylmercaptosubstituent, and a halogen substituent.
 4. The substrate of claim 2,wherein only one ligand of ligands R₅ and R₇ has a structural formula-L-W—B.
 5. The substrate of claim 2, wherein only ligand R₇ has astructural formula -L-W—B.
 6. The substrate of claim 2, comprising anasymmetric cyanine dye having a structural formula,

wherein: n is selected from 0, 1, 2 and 3; n′ is selected from 1, 2, 3,4 and 5; m′ is selected from 0 and 1; m″ is 1 when m′ is 0; m″ isselected from 0, 2, 3 and 4, when m′ is 1; X comprises O or S; eachligand of ligands R₈ and R₉ comprises H; or ligand R₈ and ligand R₉, incombination, form a fused benzene ring.
 7. The substrate of claim 1,wherein B comprises a peptide that comprises at least two enzymaticallyremovable negative charges.
 8. The substrate of claim 7, wherein atleast one enzymatically removable negative charge of the at least twoenzymatically removable negative charges is from a modifying group thatis covalently attached to B.
 9. The substrate of claim 1, wherein Bcomprises a peptide that comprises a fluorescence quencher, wherein thefluorescence quencher is sufficient for removal from the dye uponenzymatic transformation of the substrate.
 10. The substrate of claim 2,comprising a covalent bond involving B and W, wherein B comprises anenzyme substrate moiety for a β-lactamase enzyme having a structuralformula,

wherein: A comprises a fluorescence quencher, a substituted C1 to C20alkyl, or a substituted C1 to C20 aryl, optionally comprising at leastone hetero atom, and optionally comprising at least one negativelycharged group; and c is selected from 0, 1 and
 2. 11. The substrate ofclaim 1, wherein B comprises an enzyme substrate moiety for a caspaseenzyme.
 12. The substrate of claim 6, wherein n is selected from 0, 1and 2; and m″ is 2, when m″ is
 1. 13. The substrate of claim 11, whereinB comprises an enzyme substrate moiety for a caspase enzyme selectedfrom Ac-DEVD (SEQ ID NO: 1)-; Ac-LEED (SEQ ID NO: 3)-; Ac-LEND (SEQ IDNO: 7)-; Ac-VDVAD (SEQ ID NO: 8)-; AcVEID (SEQ ID NO: 2)-; Ac-IETD (SEQID NO: 9)-; and Suc-YVAD (SEQ ID NO: 10)-.
 14. The substrate of claim 1,wherein B comprises an enzyme substrate moiety for a membrane-boundenzyme, wherein the enzyme substrate moiety is hydrophobic.
 15. Thesubstrate of claim 14, wherein the substrate moiety comprises GGVVIATVK(SEQ ID NO: 11).
 16. The substrate of claim 9, wherein the fluorescencequencher is attached to the N-terminal of an enzyme substrate moiety fora caspase enzyme.
 17. The substrate of claim 1, wherein B comprises aglycosidyl.
 18. The substrate of claim 1, wherein B comprises aβ-D-glucuronidyl or a β-D-galactopyranosidyl.
 19. The substrate of claim1, wherein B comprises an enzyme substrate moiety selected from anenzyme substrate moiety for a peptidase enzyme, an enzyme substratemoiety for a histone deacetyltransferase enzyme, an enzyme substratemoiety for phosphatase enzyme, an enzyme substrate moiety for asulfatase enzyme, an enzyme substrate moiety for an esterase enzyme, anenzyme substrate moiety for a cytochrome P450 enzyme, an enzymesubstrate moiety for a glydosidase enzyme, an enzyme substrate moietyfor a phospholipase C enzyme, an enzyme substrate moiety for aphosphodiesterase enzyme, an enzyme substrate moiety for a ribonucleaseenzyme, and an enzyme substrate moiety for a β-lactamase enzyme.
 20. Thesubstrate of claim 1, wherein B comprises an enzyme substrate moietyselected from an amino acid, a peptide, an α-amino-protectedε-N-acetyllysine, a phosphoryl, a sulfuryl, a carbonyl, an alkyl, aβ-D-glucuronidyl, a β-Dgalactopyranosidyl, an α-D-galacto-pyranosidyl,an α-D-mannopyranosidyl, an α-D-glucopyranosidyl, aβ-D-glucopyrano-sidyl, a X-β-D-cellobiosidyl, aN-acetyl-β-D-galactosaminidyl, a N-acetyl-β-D-glucosaminidyl, aphosphatidylinositol, an adenosine-5′-phosphate, and anucleoside-3′-phosphate.
 21. A kit for detecting presence, absence, oractivity of an enzyme comprising: an inhibitor of activity of theenzyme; optionally, a promoter of activity of the enzyme; and asubstrate of claim 1 for the enzyme.
 22. A kit for determining an effectof a substance on an enzyme comprising: an inhibitor of activity of theenzyme; and a substrate of claim 1 for the enzyme.
 23. A method fordetecting presence, absence, or activity of an enzyme in a cell-freesystem comprising using a substrate of claim
 1. 24. A method fordetecting presence, absence, or activity of an enzyme in a live cellsystem comprising using a substrate of claim
 1. 25. A method fordetecting presence, absence, or activity of an enzyme in a living animalcomprising using a substrate of claim
 1. 26. A method for determining aneffect of a substance on an enzyme comprising using a substrate ofclaim
 1. 27. The substrate of claim 1, wherein B comprises an enzymesubstrate moiety for a peptidase enzyme.
 28. The substrate of claim 1,wherein B comprises an enzyme substrate moiety for an enzyme selectedfrom a caspase, a trypsin, a chymotrypsin, an elastase, a cathepsin B,and a cathepsin L.
 29. The substrate of claim 1, wherein ligand R₈ andligand R₉, in combination, form a fused aromatic ring.
 30. The substrateof claim 1, wherein R₆ is H.
 31. The substrate of claim 1, wherein thedotted line represents a fused benzene ring and R₄ is H.
 32. Thesubstrate of claim 1, wherein R₅ is methyl.
 33. The substrate of claim1, wherein R₇ comprises a B moiety which is an enzyme substrate moietyfor a caspase enzyme.